Pest-resistant plants comprising a construct encoding a vacuole targeting sequence and avidin or streptavidin

ABSTRACT

This invention relates to nucleic acids encoding chimeric polypeptides comprising vacuole targeting sequences and sequences encoding avidin or streptavidin. The nucleic acids are useful for conferring pest resistance on plants and in the production of compositions useful as pesticides.

FIELD OF THE INVENTION

This invention relates to chimeric polypeptides comprising vacuoletargeting sequences and plant-noxious sequences and especially pestcontrol proteins. The polypeptides are useful in methods for targetingnon-vacuolar harmful proteins to plant vacuoles. Chimeric polypeptidesof the invention containing pest control proteins are useful forconferring pest resistance on plants and in the production ofcompositions useful as pesticides. The methods and compositions formfurther aspects of the invention.

BACKGROUND OF THE INVENTION

Expression of proteins in plants is a useful strategy, for producingcommercial quantities of a desired protein. Plant expression may avoidproblems associated with production of those proteins in animal systemsparticularly where the protein is required for human therapeuticpurposes, and can also be useful for conferring beneficial properties onthe plant expression same. Such beneficial properties may includeherbicide or pest resistance for example.

However, proteins desirable for expression in plants may themselves benoxious to the plant. That is, they may harm the plant by killing ordamaging it or interfering with growth, development and fertility. Forexample, the protein avidin has been shown to cause male sterility whenexpressed in plants (WO 96/40949 and WO 99/04023), as has ribonucleasewhen used under specific promoters (Mariani et al., Symp. Soc. Exp.Biol. 45:271-9, 1991).

Accordingly, there is a need for a means of producing desirableplant-noxious proteins in a plant. Organelle targeting of proteins hasbeen contemplated (U.S. Pat. No. 5,792,923). Targeting of foreignproteins to vacuoles has also been contemplated. Vacuole targeting hasbeen applied to increasing accumulation in vacuoles of products whichwould otherwise be metabolised. U.S. Pat. No. 5,436,394 discussestargeting of invertase to the vacuole as does WO 92/14832. U.S. Pat. No.5,792,923 discloses plants in which a polyfructan sucrase is targeted tovacuoles. In U.S. Pat. No. 5,723,764 cellulose synthase is targeted tovacuoles. None of these products are plant-noxious. Accordingly, thereis no suggestion in any of these documents that vacuole targeting isrequired to avoid harmful effects on plants.

U.S. Pat. No. 5,360,726 and U.S. Pat. No. 5,525,713 contemplate vacuolartargeting of cereal lectins in leaves and other tissues. Lectins arethemselves vacuolar proteins normally located in root tips of adultplants, and specific cells of developing embryos. Lectins areinsecticidal proteins. However, there is no suggestion in any of theseU.S. parents that vacuolar targeting is necessary or advantageous forproduction of insecticidal plants.

In WO 98/11235 it is suggested that cellulose degrading enzymes betargeted to vacuoles of transgenic plants to alleviate toxicityproblems. However, no data is presented on cellulase activity orlocalisation of the protein in transgenic plants. Accordingly, there isno data showing vacuolar accumulation occurred and that toxicity wasavoided. Therefore, there is still a need for production of transgenicplants in which plant-noxious proteins can be produced withoutdeleterious effects on the plant.

One significant economic area of interest is the use of transgenicplants for pest control.

Pests such as insects, nematodes and mites are a significant economiccost to plant-based industries. Losses arise through production lost topest consumption, spoilage and introduction of disease carried by pests.

Traditionally, control of pests has been pursued though the applicationof pesticidal chemicals. Continued use of chemicals is subject to anumber of disadvantages. Pests can develop tolerance to chemicals overtime producing pesticide resistant populations. Chemical residues mayalso pose environmental hazards as well as health concerns.

Biological control presents an alternative means of pest control whichis potentially more effective and specific than current methods, as wellas reducing dependence on chemical pesticides. The need for biologicalcontrols has lead to the use of recombinant DNA techniques to insertgenes which express pesticidal toxins into plant cells.

This technology in turn may also give rise to resistant pestpopulations. There is therefore an ongoing need to find proteins withpesticidal properties, particularly those that are encoded by singlegenes. These genes can be used to transform plants to produce pestresistant cultivars.

Genes studied to date include a range of cry genes from the bacteriumBacillus thuringiensis (Bt) encoding β-endotoxins and various his-herplant genes encoding antimetabolites such as protease and α-amylaseinhibitors and lectins (Boulter, 1993). Many transgenic cultivars withimproved insect resistance are now being commercialised, for example,transgenic cotton, corn, and potatoes (James and Krattiger, 1996).

The commercial production of avidin from reproductive tissue of plantsusing such constructs has also been contemplated (U.S. Pat. No.5,767,379). The production methods are subject to a number of drawbacks.Male fertility in plants can be lost and expression in vegetative tissuemay be low. This may be due in part to expression being outside thecell.

Most recently, the use of avidin and streptavidin as larvicides againstinsect pests has been explored (WO 94/00992; Morgan et al. 1993; andBruins et al., Insect Biochemistry, 21: 535-539, 1991). In WO 94/00992generation of resistant plants has been sought by inserting into thecells of a plant a gene whose expression causes production of one ormore of those glycoproteins in larvicidal amounts. While transientexpression was shown in maize cells in suspension, no data is presentedto show that avidin or streptavidin were expressed at insecticidalconcentrations or that plants could be produced expressing same withoutdeleterious side effects.

In later applications by the same applicant as for WO 94/00992,transgenic plants with avidin under control of a promoter are described,see WO 96/40949, WO 99/04023 and U.S. Pat. No. 5,767,379. There is nomention of any of the plants produced in these documents as havinginsecticidal activity. Moreover, the plants produced all exhibit malesterility. There is no specific suggestion in these documents thatvacuole targeting could be used to avoid development of male sterility.Similarly, in Plant Physiol. 102 (Suppl.): 45, 1993 a chimeric genecomprising streptavidin coding sequences under control of the CaMV 35Spromoter and three signal sequences is contemplated. The signalsequences are indicated as useful for targeting protein to differentorganelles in plants. However, these organelles are unspecified.Moreover, there is no evidence any plants have been producedincorporating the chimeric genes nor any discussion as to the effectsthe genes may have on those plants.

The issues with chimeric genes is whether they can be correctlytargeted, whether they will be stable in vacuoles, and whethersequestration in a cell vacuole will prevent the protein expressed bythe chimeric gene from having deleterious effects on the plant cells.

To date, limited success has been achieved in producing insect resistantplants using this technology.

Specifically, no one has been able to produce a fertile plant expressingsignificant levels of a biotin-binding protein in vegetative tissues,nor plants shown to be resistant to insect attack due to the expressionof a biotin-binding protein. Similarly, no one has yet been able toprovide a protein conferring broad spectrum insect resistance on a hostplant without deleterious effects to the plant.

It is an object of the present invention to provide chimericpolypeptides and plants which go some way to overcoming the abovedrawbacks or at least to provide the public with a useful choice.

SUMMARY OF THE INVENTION

Accordingly, in one aspect, the present invention may be broadly said toconsist in a chimeric polypeptide that comprises (a) a vacuole targetingsequence encoding a polypeptide; and (b) a sequence encoding aplant-noxious protein linked in operable combination to said targetingpolypeptide.

Preferably, the vacuole targeting polypeptide is a signal sequencepolypeptide selected from proteinase inhibitor signal sequence (PPI-I orPPI-II) polypeptide which have the amino acid sequences set out in FIG.8B and FIG. 9B respectively, or variants thereof having substantiallyequivalent signalling activity thereto.

Preferably, the plant-noxious protein is pest control protein anddesirably, a biotin-binding protein.

Preferably, the biotin-binding protein encoded is avidin or streptavidinor a functionally equivalent variant thereof.

The chimeric polypeptides may further comprise at least one additionalsequence encoding a protein or peptide.

Conveniently, the chimeric polypeptides of the invention are obtained byexpression of a DNA sequence encoding the chimeric polypeptide in a hostcell or organism.

In a further aspect, the present invention provides an isolated nucleicacid molecule encoding a chimeric polypeptide of the invention.

This nucleic acid molecule can be an RNA or cDNA molecule but ispreferably a DNA molecule.

Also provided by the present invention are recombinant expressionvectors which contain a DNA molecule of the invention, and hoststransformed with the vector of the invention capable of expressing apolypeptide of the invention.

In a still further aspect, the invention provides a method of producinga polypeptide of the invention comprising the steps of:

-   (a) culturing a host cell which has been transformed or transfected    with a vector as defined above to express the encoded polypeptide of    the invention; and optionally-   (b) recovering the expressed polypeptide.

An additional aspect of the present invention provides a ligand thatbinds to a polypeptide of the invention. Most usually, the ligand is anantibody or antibody binding fragment.

In a further aspect the present invention provides a method forproducing a pest resistant plant, comprising transforming the plantgenome to include at least one DNA molecule of the invention whichincludes a sequence encoding a pest control protein.

Also provided is a transgenic plant expressing insecticidally effectiveconcentrations of a pest control protein.

The present invention further provides a transgenic plant that containsa DNA molecule of the invention.

In one embodiment the transgenic plant further contains at least oneadditional DNA sequence encoding a protein or peptide.

In a still further aspect, the present invention provides a method forcontrolling or killing pests comprising administering to said pest anamount of a chimeric polypeptide of the invention, which includes asequence encodine a pest control protein, effective to control or killsaid pest.

In one embodiment of the method, the chimeric polypeptide isadministered with a second pest control protein, where the combinationprovides more effective control than administration of the second pestcontrol protein alone.

Usually, the pests are the immature stages of insects, including larvae,grubs, nymphs and instars.

In yet a further aspect, the present invention provides a compositioncomprising a chimeric polypeptide of the invention and a carrier,diluent, excipient or adjuvant.

In a further composition aspect, the present invention provides acomposition comprising plant material produced in accordance with theinvention and a carrier, diluent, excipient or adjuvant.

The composition is preferably a pesticidal composition.

In a further aspect the present invention provides a method forcontrolling or killing pests comprising administering to said pest plantmaterial produced in accordance with the invention, which expresses apest control protein, or administering a pesticidal composition of theinvention, effective to control or kill said pest.

In a still further aspect the present invention provides a method forproducing a plat-noxious protein, the method comprising extracting theprotein from a plant containing a DNA molecule of the invention codingfor same.

While the invention is broadly as defined above, it will be appreciatedby those persons skilled in the art that it is not limited thereto andthat it also includes embodiments of which the following descriptiongives examples.

FIG. 1 shows the nucleic acid sequence of Potato Proteinase Inhibitor I(PPI-I/pUC19)(SEQ ID NO:1). The signal sequence is in bold type and thestart and stop codons are in italic. The mutagenic primer is denoted byunderlined in lower case with the Bgl II site created by mutagenesis inbold italic. The upstream and downstream primers used were the Forwardand Reverse M13(lacZ) Primers [Perkin Elmer].

FIG. 2 shows Avidin cDNA (pGEMav) (SEQ ID NO:2. The signal sequencerepresented in bold type, start and stop codons are in italic, primersare underlined lower case with the BamH I site created by mutagenesis initalic. The downstream primer used was the Reverse M13(lacZ) Primer[Perkin Elmer].

FIG. 3 shows streptavidin cDNA (Streptavidin/pUC19) (SEQ ID NO:3). Startand stop codons are in bold type. EcoR I and Xba I sites are in italic.

FIG. 4 shows potato proteinase inhibitor II (PPI-II/pUC19) (SEQ IDNO:4). The signal sequence is represented in bold type and start andstop codons are in bold italic. Underlined type denotes the intronwithin the signal sequence. The asterisk denotes the result of PCR errorduring isolation of the PPI-II sequence.

FIG. 5 shows components of the ligation reaction to produce recombinantpART7 containing the PPI-I signal sequence/Avidin cDNA gene fusion. A)PPI-I leader fragment resulting from a Sal I/Bgl II digest of themutated PPI-I PCR product. B) Avidin mature protein cDNA fragment,resulting from a BamH I/Hind III digest of the mutated Avidin PCRproduct. C) pART7 vector following an Xho I/Hind III digestion. *denotes compatible cohesive ends. ** denotes compatible cohesive ends.

FIG. 6 shows DNA fragments A, B and C were the components of theligation reaction to produce recombinant pUC19 containing the PPI-IIsignal sequence/Streptavidin cDNA gene fusion. The fused gene was thenreleased from pUC19 by a Sal I/BamH I digest and ligation of componentsD and E produced recombinant pART7. A) PPI-II leader fragment resultingfrom a Sal I/EcoR I digest of the PPI-II PCR product. B) StreptavidincDNA fragment, resulting from an EcoR I/Xba I digest of the recombinantplasmid pUC19/Streptavidin cDNA. D) PPI-II signal sequence/StreptavidincDNA gene fusion fragment, resulting from a Sal/BamH I digest ofrecombinant pUC19 containing the fused gene. E) pART7 vector followingan Xho I/BamH digestion. * denotes compatible cohesive ends.

FIG. 7 shows a schematic representation of the pART7 expression cassetteas it was cloned into the pART27 binary vector; A) containing thePPI-I-Avidin gene fusion and B) containing the PPI-II/Streptavidin genefusion (altered BamH I site=SEQ ID NO:5).

FIG. 8 shows PPI-I/Avidin gene fusion sequence (SEQ ID NO:6) (A) andfusion protein sequence (SEQ ID NO:7)(B): The fusion protein has a totalof 161 amino acids; the PPI-I sequence is represented by italic typewith bold type denoting the PPI-I signal peptide. Two amino acids, novelto both the PPI-I and the Avidin peptide sequences and represented inlower case, were introduced with the ligation of the Bgl II and BamH Icompatible cohesive ends.

FIG. 9 shows PPI-II/Streptavidin gene fusion sequence (SEQ ID NO:8)(A)and fusion protein sequence (SEQ ID NO:9) (B): The fission protein has atotal of 168 amino acids; the PPI-II sequence is represented by italictype with bold type denoting the PPI-II signal peptide. Three aminoacids, novel to both PPI-II and the Streptavidin peptide sequences andrepresented in lower case, were introduced at the point of fusion.

FIGS. 10 and 11 show the survival of larvae of the potato moth,Phthorimaea operculella fed tobacco plants expressing avidin in tworeplicate trials.

FIG. 12(A) shows the nucleotide sequence for the gene for streptavidin(SEQ ID NO:10)(Argarana et al. 1986). The signal sequence is representedin bold type, start and stop codons in bold italic. (B) shows theprotein sequence for streptavidin (SEQ ID NO:11). The signal sequence isrepresented in bold type.

FIG. 13 shows a cross section of a transgenic leaf stained withmethylene blue/Azure II to show general structure of the leaf. Denselystained bodies in the vacuole are arrowed. Bar=50 μm. v; vascularbundle. t; trichome. g; glandular hair.

FIG. 14 shows immunolabelling of the section for the distribution ofavidin (arrowed). Fluorescence indicates the presence of avidin. Bar=50μm.

FIG. 15 shows a transmission electron micrograph showing thedistribution of protein bodies in the vacuole of the cell (arrowed).Bar=1 μm.

FIG. 16 shows a higher magnification of FIG. 15. Immunogold labellingover the surface of the protein bodies within the vacuole (arrowed).Bar=200 nm.

FIG. 17 shows the survival of larvae of the potato tuber moth,Phthorimaea operculella fed tobacco plants expressing streptavidin intwo replicate trials.

FIG. 18 shows the proportion of larvae of the potato tuber moth,Phthorimaea operculella at each instar after feeding for nine daystobacco plants expressing streptavidin.

FIG. 19 shows the growth of larvae of the common cutworm, Spodopteralitura, fed tobacco leaves expressing avidin.

FIG. 20 shows the survival of larvae of the common cutworm, Spodopteralitura, fed tobacco leaves expressing avidin.

FIG. 21 shows the accumulation of larval biomass of the common cutwormSpodoptera litura, fed tobacco leaves expressing avidin.

FIG. 22 shows the growth of larvae of the cotton bollworm (corn earworm,tomato fruitworm), Helicoverpa armigera fed tobacco leaves expressingavidin.

FIG. 23 shows the survival of larvae of the cotton bollworm (cornearworm, tomato fruitworm), Helicoverpa armigera, fed tobacco leavesexpressing avidin.

FIG. 24 shows the accumulation of biomass of larvae of the cottonbollworm (corn earworm, tomato fruitworm), Helicoverpa armigera, fedtobacco leaves expressing avidin.

FIG. 25 shows the effect of the level of avidin expression in tobacco onthe growth of larvae of the cotton bollworm (corn earworm, tomatofruitworm), Helicoverpa armigera.

FIG. 26 shows the effect of the level of avidin expression in tobacco onthe survival of larvae of the cotton bollworm (corn earworm, tomatofruitworm), Helicoverpa armigera.

FIG. 27 shows the effect of the level of avidin expression in tobacco onthe accumulation of biomass of larvae of the cotton bollworm (cornearworm, tomato fruitworm), Helicoverpa armigera.

FIG. 28 shows the effect of avidin and streptavidin incorporated intoinsect diet at three concentrations on the growth of larvae of the pineshoot tip moth, Rhyacionia buoliana.

FIG. 29 shows the effect of avidin and streptavidin incorporated intoinsect diet at three concentrations on the survival of larvae of thepine shoot tip moth, Rhyacionia buoliana.

FIG. 30 shows the effect of avidin and streptavidin incorporated intoinsect diet at three concentrations on the accumulation of biomass oflarvae of the pine shoot tip moth, Rhyacionia buoliana.

FIG. 31 shows the effect of avidin-painted willow leaves on the survivalof larvae of the willow sawfly, Nematus oligospilus.

FIG. 32 shows the effect of avidin-painted willow leaves on the weightgain of larvae of the willow sawfly, Nematus oligospilus.

FIG. 33 shows the effect of avidin-painted willow leaves of theformation on the pupae of the willow sawfly, Nematus oligospilus.

FIG. 34 shows the effect of avidin-painted willow leaves on theemergence of adults of the willow sawfly, Teleogryllus commodus.

FIG. 35 shows the effect of avidin-painted lettuce leaves the growth ofnymphs of the black field cricket, Teleogryllus commodus.

FIG. 36 shows the effect of avidin-painted lettuce leaves on thesurvival of nymphs of the black field cricket, Teleogryllus commodus.

FIG. 37 shows the effect of avidin-painted lettuce leaves on theaccumulation of biomass of nymphs of the black field cricket,Teleogryllus commodus.

FIG. 38 shows the effect of streptavidin incorporated into insect dieton the survival of neonate larvae of the clover root weevil, Sitonalepidus.

FIG. 39 shows the effect of streptavidin incorporated into insect dieton the survival of larvae of the Argentine stem weevil, Listronotusbonariensis.

FIG. 40 shows the effect of avidin-painted clover leaves on the survivalof adults of the clover root weevil, Sitona lepidus.

FIG. 41 shows the effect of avidin added to pollen on the consumption ofthat food by adult honeybees, Apis mellifera.

FIG. 42 shows the effect of avidin added to pollen on the survival ofadult honeybees. Apis mellifera.

FIG. 43 shows the effect of avidin-painted lettuce leaves on the weightsof snails, Cantareus aspersus.

FIG. 44 shows the effect of avidin-painted lettuce leaves on thesurvival of snails, Cantareus aspersus.

FIG. 45 shows the effect of avidin-painted lettuce leaves on the weightof slugs, Deroceras reticulatum.

FIG. 46 shows the effect of avidin-painted lettuce leaves on thesurvival of slugs, Deroceras reticulatum.

FIG. 47 shows the effect of avidin expression in tobacco combined withpainted-on aprotinin or Cry1Ba on survival of larvae of the cottonbollworm (corn earworm, tomato fruitworm), Helicoverpa armigera.

FIG. 48 shows the effect of avidin expression in tobacco combined withpainted-on aprotinin on growth of larvae of the cotton bollworm (cornearworm, tomato fruitworm), Helicoverpa armigera.

FIG. 49 shows the effect of avidin expression in tobacco combined withpainted-on aprotinin on biomass of larvae of the cotton bollworm (cornearworm, tomato fruitworm), Helicoverpa armigera.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel chimeric polypeptides comprisingvacuole targeting sequences and plant-noxious sequences. The targetingsequences and plant-noxious sequences are operably linked.

The term “operably linked” as used herein refers to a juxtapositionwherein the components so described are in a relationship permittingthem to function in their intended manner. For example, a signalsequence is operably linked to a coding sequence if the promoter affectsits transcription or expression.

The term “vacuole targeting sequence” as used herein refers to asequence operable to direct or sort a selected non-vacuolar protein towhich such sequence is linked, to a plant vacuole.

The vacuolar targeting polypeptide sequences of the invention, whentransformed into plants, function to direct or sort the protein productsdirected by the expression of genes to which they are operably linkedfrom the cytoplasm to the vacuole of the plant cell. Since the vacuoleof plant cells has a storage function, proteins directed there remainthere, continually increasing in abundance, unless subject todegradation by vacuolar proteinases. The vacuolar proteins are alsoisolated from the major metabolic processes in the plant and thus willnot interfere with the plant growth and development. The success of thepresent invention needed that both these requirements be met.

Vacuolar targeting sequences include any such targeting sequences as areknown in the art that effect proper vacuole targeting in plant hosts.These include polypeptides targeting barley lectin (Bednarek et al.,1990), sweet potato sporarnin (Matsuoka et al., 1990), tobacco chitinase(Neuhaus et al., 1991), bean phytohemagglutinin (Tague et al. 1990), 2Salbumin (Saalbach et al., 1996), aleurain (Holwerda et al., 1992).Vacuolar targeting in plants has been widely studied (for example seeChrispeels, 1991; Chrispeels & Raikhel, 1992; Dromboski & Raikhel, 1996;Kirsch et al., 1994; Nakamura & Matsuoka, 1993; Neilsen et at., 1996;Rusch & Kendall, 1995; Schroder et al., 1993; Vitale & Chrispeels, 1992;von Heijne, 1983). Other sequences are described, for example, in U.S.Pat. No. 5,436,394, U.S. Pat. No. 5,792,923, U.S. Pat. No. 5,360,726,U.S. Pat. No. 5,525,713 and U.S. Pat. No. 5,576,428 incorporated hereinby reference. However, potato proteinase inhibitor targeting sequencesare preferred.

A number of potato proteinase signal sequence polypeptides designatedPPI-I and PPI-II are disclosed for use herein. These polypeptides weredescribed previously (Beuning et al., (1994); Christeller et al.(1994)). The polypeptides have the amino acid sequences set out in FIGS.8B and 9B respectively. Also encompassed within the invention arevariants of these polypeptides and those known in the art which havesubstantially equivalent targeting sequence activity thereto.

The term “variant” as used herein refers to a polypeptide wherein theamino acid sequence exhibits substantially 70% or greater homology withthe amino acid sequences set out in FIGS. 1 and 4. Preferably, thevariants will have greater than 85% homology, and most preferably, 95%homology or more. Variants may be arrived at by modification of thenative amino acid sequence by such modifications as insertion,substitution or deletion of one or more amino acids.

As noted above, the chimeric polypeptide comprises a vacuole targetingsignal sequence operably linked to a plant-noxious protein.

The term “plant-noxious protein” as used herein refers to a proteinwhich has a negative effect on plant health, growth, development orfertility when not sequestered in a plant vacuole.

Examples of plant-noxious proteins include barnase (ribonuclease),cellulases and other cell wall degrading enzymes such as pectinases andpolygalacturonases as well as pest control proteins discussed below.

In one embodiment, the plant-noxious protein is a pest control proteinspest control proteins include proteins which decrease availability ofvitamins, or other essential growth component or are toxic to pests perse. Toxic proteins include lectins, proteinase inhibitors, Bacillusthuringiensis insecticidal proteins, alpha-amylase inhibitors,vegetative insecticidal proteins, lipoxygenase and cholesterol oxidase.Proteins which decrease availability of vitamins fall broadly into thesecategories of degradative enzymes and binding proteins. Examples ofdegradative enzymes include thiaminase, riboflavin hydrolase, andpantothenate hydrolase but are not limited thereto.

Bt proteins useful in the present invention include Cry proteins such asCry1Ba, Cry1Ac, Cry1Cb, Cry1Da, Cry1F, Cry5 and Cry9A, but are notlimited thereto.

Proteinase inhibitors useful in the invention include aprotinin,kunitz-type inhibitors from soybean, arrowroot, taro, proteinaseinhibitor 1, proteinase inhibitor 2, alpha-1 antitrypsin, Bownan-Birkinhibitors from soybean and cowpea and oryzacystatin.

The term “pest” as used herein refers to a broad group of organismswhich at some point in their life cycle live or feed on plants adverselyaffecting same. Included in the term are protozoa, arthropods(especially insects), aschelminthes and platyhelminthes, nematodes andmolluscs.

Binding proteins useful in the invention include riboflavin-bindingprotein, carotenoid binding proteins, fatty-acid binding proteins,retinol binding proteins, alpha-tocopherol binding proteins,folate-binding proteins, thiamine-binding proteins, pantothenate-bindingproteins and biotin-binding proteins, but again are not limited thereto.A preferred group of binding proteins are vitamin binding proteins,particularly biotin-binding proteins. These are proteins which associatewith biotin to form a complex with a dissociation constant of 10⁻⁶M orless. Usually, the complex is a non-covalent complex. The biotin bindingproteins for use herein must be operable to bind biotin in a plantsystem without adversely affecting the plant, or to affect the plant ina minimal way, when included in chimeric to polypeptides of theinvention. For example, slight reductions in plant growth would beacceptable.

Systems requiring covalent enzymatic sequestration are also contemplatedwithin this term. For example, simultaneous overexpression of a biotinrequiring carboxylase or a biotin acceptor peptide (for example, seeSchatz, P. J., Biotechnology, 11: 1138-1143, (1993) and biotinholocarboxylase synthetase in the vacuole could be used to induce biotindeficiency. Biotin would be covalently sequestered enzymatically onvacuole rupture.

Biotin is an essential nutrient for many species of pests (Dadd R. H.,1985; Kerkut G. A. et al., Comprehensive Insect Physiology, Biochemistryand Pharmacology, 4: 313-390, 1985). As discussed above, biotin-bindingproteins have been found to have pesticidal properties and to inhibitgrowth of pests. The binding of biotin causes a biotin deficiency whichresults in the inhibition of growth and ultimate death of pests.

Biotin-binding proteins known in the art include egg yolk biotin-bindingproteins (Subramanial and Ariga 1995, Biochem J, 308: 573-577, serum(Seshagiri and Ariga, 1987. Biochem. Biophys. Acta, 916: 474431),biotin-binding antibodies, and fragments thereof, biotin holocarboxylasesynthetase, biotinidase, bacterial proteins, avidin isolated from eggwhite, and streptavidin. The properties of a number of these proteinsare usefully discussed in Methods of Enzymology Vol 184 (eds M. Wilchetaand E A Bayer).

Preferred biotin-binding polypeptides, for use in the present invention,are avidin and streptavidin or functionally equivalent variants thereof.It will be appreciated that other groups that function to bind biotin,such as those referred to above, are equally able to be used in thepresent invention.

Avidin is a water-soluble tetrameric glycoprotein isolated originallyfrom raw egg white (J. Biol. Chem 136: 801 (1940)). The protein is wellknown with the complete amino acid sequence having been published in,for example, J. Biol. Chem. 246: 698 (1971).

The full amino acid sequence for avidin is shown in FIG. 8B (amino acids34 to 161). Several natural variants of avidin have also been discussedin Keinanen et al., Eur. J., Biochem, 220:615-621 (1994) and syntheticvariants in Marttila et al., FEBS Letters. 441:313-317 (1998).

Streptavidin is a non-glycosylated bacterial binding protein derivedfrom the culture supernatant of Streptomyces avidinii (Bayer et al.1990). The full amino acid sequence for streptavidin is given in FIG.12.

‘Core’ SAV is equivalent to amino acid residues 37-164 of Streptomycesavidinii (SAV) FIG. 12, (Argarana et al., 1986). Other ‘core’ SAVmolecules have been produced with various N-terminal and C-terminaldeletions. A preferred sequence referred to as “Synthetic ‘Core’Streptavidin” is a modified ‘core’ SAV having the sequence shown in FIG.9B (amino acids 41 to 168). SYNSAV is equivalent to ‘Core’ SAV modifiedsuch that codons for each amino acid correspond to those in highlyexpressed E. coli genes. SYNSAV is also modified to contain uniquerestriction sites evenly throughout sequence. The resulting sequence hasG+C content of 54% relative to 69% for same region of native SAV(Thompson et al. (1993))²⁹. A number of natural variants of streptavidinhave also been described in Bayer et al., Biochem. Biophy. Acta 1263:60-66 (1995), GenBank Acc. No. S78782 and S78777. Synthetic streptavidinmolecules can also be produced using known art techniques. See forexample WO 89/03422.

The chimeric polypeptides of the invention may further comprise one ormore sequences encoding other proteins or peptides. Two to four furthersequences are contemplated, but more are feasible. These other proteinsor peptides may be selected from any proteins known in the art which itis desired to express in a plant vacuole including plant-noxiousproteins discussed above.

Proteins to be produced in conjunction with pest control proteins may beselected so as to achieve an additive or synergistic effect asdemonstrated in Example 18), a broader spectrum of control, or to reducethe risk of resistance developing. Examples of such proteins includeother pest control proteins as discussed above including proteinaseinhibitors, toxic proteins and biotin-binding proteins, as well asantimicrobial, antifungal and antiviral proteins but not limitedthereto.

The applicants have surprisingly found that plants expressing avidinwhen combined with Bt insecticidal protein can exhibit synergisticeffects on pests (FIG. 47). Proteinase inhibitors may be desirable foruse in preventing proteolysis of the insect control protein (see Example18) Shao et al., J Invertebr. Pathol. 72: 73-81 (1998); and Keller etal., Insect Biochem. Mol. Biol. 26: 365-73 (1996). The compatibility ofbiotin-binding proteins and protease inhibitors has been demonstrated bythe applicant.

The antimicrobial, antifungal and antiviral groups of proteins canassist in the control of plant disease particularly where insect damagecontributes to the spread of disease. Proteins which have been shown tohave these activities include dermaseptins, cercropins, attacins,lysozyme, chitinases, hevein, glucose oxidase, glucanases, thionins,lectins, Raphanus sativus, antifungal protein, osmotin, lipid transferproteins, lipoxygenase and virus coat proteins.

Similarly, reduction in disease from insect resistant crops has beenreported. For example, research at Iowa State University has shownreduction in feeding damage is linked to a reduction in earmould in Btmaize.

The reader will appreciate that modifications, including chemical andbiochemical modifications, of the polypeptides of the invention arepossible. Such modifications include, for example, acetylation,carboxylation, phosphorylation, glycosylation, ubiquitination,labelling, and the like. The production of peptide fragments is alsowell within the capabilities of an art skilled worker.

The polypeptides of the invention can be prepared in a variety of ways.For example, as indicated above the signal sequences and biotin-bindingproteins can be produced by isolation from natural sources and thencoupled using techniques known in the art. For example, throughrecombinant nucleic acid methods.

Synthesis using known techniques (such as stepwise solid phase synthesisdescribed by Merryfield, J. Amer. Chem.Soc. Vol 85:2149-2156, 1963), oras preferred through employing recombinant DNA techniques.

Variants of the polypeptide can similarly be made by any of thosetechniques known in the art. For example, variants can be prepared bysite-specific mutagenesis of the DNA encoding the native amino acidsequence as described by Adelman et al. DNA 2:183 (1983). Generally, thevariants produced are functionally equivalent to the original sequence.

Where it is preferred, recombinant techniques used to produce thepolypeptide of the invention, the first step is to obtain DNA encodingthe desired product. Such DNA comprises a still further aspect of thisinvention.

The DNA of the invention may encode a native or modified polypeptide ofthe invention or an active fragment thereof. In its presently preferredform, the DNA comprises the nucleotide sequence of FIG. 8A, or thenucleotide sequence of FIG. 9A. Preferred sequences exhibit 60% orgreater homology with these sequences, preferably 80% homology and mostpreferably 95% homology or more. That is, most preferred sequences willhybridise to the sequences of the invention under stringenthybridisation conditions.

The DNA can be isolated from any appropriate natural source or can beproduced as intron free cDNA using conventional techniques. DNA can alsobe produced in the form of synthetic oligonucleotides where the size ofthe active fragment to be produced permits. By way of example, theTriester method of Matteucci et al. J. Am. Chem.Soc. Vol 103:3185-3191(1981) may be employed.

Where desirable, the DNA of the invention can also code for a chimericpolypeptide of the invention (including polypeptides encoding more thanone protein). Such fusion proteins may be produced as disclosed in WO86/02077 incorporated herein by reference. Fusion proteins furthercomprising the polypeptide of the invention and a carrier protein arepossible. This carrier protein will generally be cleavable from thepolypeptide, peptide or fragment under controlled conditions. Examplesof commonly employed carrier proteins are galactosidase andglutathione-S-transferase.

As indicated above, also possible are variants of the polypeptide orpeptide which differ from the native amino acid sequence by insertion,substitution or deletion of one or more amino acids. Neutral variations(those which have no effect on function) are specifically contemplated.Where such a variant is desired the nucleotide sequence of the nativeDNA is altered appropriately. This alteration can be made throughelective synthesis of the DNA or by modification of the native DNA by,for example, site-specific or cassette mutagenesis. Preferably, whereportions of cDNA or genomic DNA require sequence modifications,site-specific primer directed mutagenesis is employed using techniquesstandard in the art.

In a further aspect, the present invention consists in replicabletransfer vectors suitable for use in preparing a polypeptide or peptideof the invention. These vectors may be constructed according totechniques well known in the arL or may be selected from cloning vectorsavailable in the art.

The cloning vector may be selected according to the host or host cell tobe used. Useful vectors will generally have the followingcharacteristics:

-   (a) the ability to self-replicate;-   (b) the possession of a single target for any particular restriction    endonuclease; and-   (c) desirably, carry genes for a readily selectable marker such as    antibiotic resistance or herbicide tolerance.

Two major types of vector possessing these characteristics are plasmidsand bacterial viruses (bacteriophages or phases). Presently preferredvectors include the plasmids pMOS-Blue, pGem-T, pUC18, pUC19, pART27,pMON, pJIT, pBIN, pRD 400, pART7.

Also contemplated is the use of RNA vectors for example, tobacco mosaicvirus (Donson et al., Proc Natl. Acad. Sci. USA., 88:7204-8, 1991),potato virus X (PVX)(Chapman et al., Plant J. 2:549-57, 1992), andbarley stripe mosaic virus (ESMV) (Josh, et al., EMBO J. 9:2663-9,1990). TMV has previously been used to infect plants to producetherapeutic protein products (Turpen, Philos Trans. R. Soc. Lond. Biol.Sci., 354: 665-73, 1999). Basic RNA vectors can be produced according toknown art techniques.

The DNA molecules of the invention may be expressed by placing them inoperable linkage with suitable control sequences in a replicableexpression vector. Control sequences may include origins of replication,a promoter, enhancer and transcriptional terminator sequences amongstothers. The selection of the control sequence to be included in theexpression vector is dependent on the type of host or host cell intendedto be used for expressing the DNA.

Generally, procaryotic, yeast, insect or mammalian cells are usefulhosts. Also included within the term hosts are plasmid vectors. Suitableprocaryotic hosts include E. coli, Bacillus species and various speciesof pseudomonas. Commonly used promoters such as β-lactamase(penicillinase) and lactose (lac) promoter systems are all well known inthe art. Any available promoter system compatible with the host ofchoice can be used. Vectors used in yeast are also available and wellknown. A suitable example is the 2 micron origin of replication plasmid.

Similarly, vectors for use in mammalian cells are also well known. Suchvectors include well known derivatives of SV-40, adenovirus,retrovirus-derived DNA sequences, Herpes simplex viruses, and vectorsderived from a combination of plasmid and phage DNA.

Further eucaryotic expression vectors are known in the art (e.g. P. J.Southern and P. Berg, J. Mol. Appl. Genet. 1 327-341 (1982); S.Subramani et al., Mol.Cell.Biol. 1, 854-864 (1981); R. J. Kaufmann andP. A. Sharp, “Amplification and Expression of Sequences Cotransfectedwith a Modular Dihydrofolate Reducase Complementary DNA Gene, J. Mol.Biol. 159, 601-621 (1982); R. J. Kaufmann and PA. Sharp, Mol. Cell.Biol. 159, 601-664(1982); S. I. Scahill et al., “Expressions AndCharacterization Of The Product Of A Human Immune Interferon DNA Gene InChinese Hamster Ovary Cells,” Proc. Natl. Acad. Sci. USA. 80, 4654-4659(1983); G. Urlaub and L. A. Chasin, Proc. Natl. Acad. Sci. USA. 77,42164220, (1980).

The expression vectors useful in the present invention contain at leastone expression control sequence that is operatively linked to the DNAsequence or fragment to be expressed. The control sequence is insertedin the vector in order to control and to rate the expression of thecloned DNA sequence. Examples of useful expression control sequences arethe lac system, the trp system, the tac system, the trc system, majoroperator and promoter regions of phage lambda, the glycolytic promotersof yeast acid phosphatase, e.g. Pho5, the promoters of the yeastalpha-mating factors, and promoters derived from polyoma, adenovirus,retrovirus, and simian virus, e.g. the early and late promoters of SV40,and other sequences known to control the expression of genes ofprokaryotic and eucaryotic cells and their viruses or combinationsthereof.

Also useful in the present invention are promoters which can be used totarget proteins to specific plant tissues. These have application insituations where accumulation of a protein in a particular tissue isdesired, or alternatively, is to be avoided to prevent non-targeteffects. For example, accumulation of an insect control protein inpollen may be undesirable if it is fed on by non target pests such asbutterflies, bees or other pollinators. Specific promoters can be usedto target such pest control proteins away from pollen.

Alternatively, a target pest may have defined feeding characteristicssuch as only feeding on leaves, seed, fruit, flowers or the like. Insuch cases, it would be desirable to target the pest control protein tothe plant tissues being feed on, or to particular cells within thosetissues. For example, to leaf epidermal cells, root cortex cells,mesophyll cells and the like. Any of the promoters known in the art fortargeting specific plant tissues may be employed.

Preferred promoters for use herein include lacZ, CaMV-35S, LHC a/b, T7,nos, rubisco small subunit (SSU), gpd and nod gene promoters.

In the construction of a vector it is also an advantage to be able todistinguish the vector incorporating the foreign DNA from unmodifiedvectors by a convenient and rapid assay. Such assays include measurablecolour changes, antibiotic resistance, herbicide tolerance and the like.In one preferred vector, the β-galactosidase gene is used, which gene isdetectable by clones exhibiting a blue phenotype on X-gal plates. Thisfacilitates selection.

Once selected, the vectors may be isolated from the culture usingroutine procedures such as freeze-thaw extraction followed bypurification.

For expression, vectors containing the DNA of the invention to beexpressed and control signals are inserted or transformed into a host orhost cell. Intermediate host cells can be used to increase the copynumber of the cloning vector prior to introduction into plant cells.Some useful expression host cells include well-known prokaryotic andeucaryotic cells. Some suitable prokaryotic hosts include, for example,E. coli, such as E. coli, S G-936, E. coli HB 101, E. coli W3110, E.coli X1776, E. coli, X2282, E. coli, DHT, and E. coli, MR01,Pseudomonas, Bacillus, such as Bacillus subtilis, and Streptomyces.Suitable eucaryotic cells include yeast and other fungi, insect, animalcells, such as COS cells and CHO cells, human cells and plant cells intissue culture.

Expression systems employing insect cells utilising the control systemsprovided by baculovirus vectors have been described (Miller et al., inGenetic Engineering, 8: 277-297, 1986).

Depending on the host used, transformation is performed according tostandard techniques appropriate to such cells. For prokaryotes or othercells that contain substantial cell walls, the calcium treatment process(Cohen, S. N. Proceedings, National Academy of Science, USA 69: 2110,1972) may be employed. For mammalian cells without such cell walls thecalcium phosphate precipitation method of Graeme and Van Der Eb,Virology 52:546, 1978 is preferred. Transformations into plants may becarried out using Agrobacterium tumefaciens (Shaw et al., Gene 23:315,1983) or into yeast according to the method of Van Solingen et al. J.Bact. 130: 946, 1977 and Hsiao et al. Proceedings, National Academy ofScience, 76: 3829, 1979.

In a preferred transformation process, the vectors of the invention areincorporated into Agrobacterium tumefaciens which can be used to infectplant cells, particularly dicotyledenous plant cells, therebytransferring the vectors and conferring pest resistance. The cloningvectors can also be introduced into plant cells using convenient arttechniques such as electroporation, microparticle bombardment andmicroinjection. Microparticle bombardment is the preferredtransformation process for monocotyledenous plants. Suitable planttransformation techniques are usefully summarised in Torres et al.,Plant Cell, Tissue and Organ Culture 34: 279-285, 1993, Michelmore etal., Plant Cell Reports 6:439-442, 1987, Horsch et al., Plant MolecularBiology Mammal AS: 1-9, 1988, Xinrun et al., J. Genet. and Breed 46:287-290, 1992 and WO 97/17455 incorporated herein by reference.

Upon transformation of the selected host with an appropriate vector thepolypeptide encoded can be produced, often in the form of fusionprotein, by culturing the host cells. The polypeptide of the inventionmay be detected by rapid assays as indicated above. The polypeptide canthen be recovered and purified if desired. Recovery and purification canbe achieved using any of those procedures known in the art, for exampleby absorption onto and elution from an anion exchange resin. This methodof producing a polypeptide of the invention constitutes a further aspectof the present invention.

The present invention also provides a method for producing aplant-noxious protein, the method comprising extracting the protein froma plant incorporating a DNA sequence of the invention coding for same.The expression level of the protein may be increased by furtherincorporating into the DNA sequence of the invention a peptide exportsignal sequence, or intron sequence. Methods of enhancing expressionlevels and methods for production of the protein generally may beeffected according to the techniques of WO 97/17455 incorporated hereinby reference.

The use of the chimeric polypeptides of the present invention representsan advance over this document because the protein is produced invegetative tissues (leaves, stems, tubers, roots) as opposed to thereproductive tissues. In the case of avidin and streptavidin this avoidsthe negative effect of male sterility.

The method of the present invention is also significantly more effectivethan the disclosed art method with avidin produced as levels up to twotimes higher than previously reported. See Examples 9, 18 and 17.

The applicant has also been the first to achieve expression ofstreptavidin using this methodology (see Example 7). The expressionlevels are approximately twice those previously reported for avidin inU.S. Pat. No. 5,767,379.

It is also noted that the avidin and streptavidin expression levels wereachieved with selfed plants. Higher levels of expression are anticipatedwhere plants expressing high levels of avidin or streptavidin arecrossed.

The method can be used for producing proteins from a wide range ofplants which produce abundant vegetative material e.g. potatoes,cassaya, tobacco, grasses, legumes, and trees rather than beingrestricted to plants which produce large reproductive structures e.g.maize.

In a further aspect, the present invention provides a transgenic plantthat contains a DNA molecule of the invention. The plant is producedaccording to the procedures detailed above, generally comprisingtransformation with a vector of the invention.

In one embodiment, the transgenic plant contains at least one, andcommonly two to four, additional DNA sequences encoding a protein orpeptide. More additional sequences are feasible. The proteins orpeptides may be any of those proteins or peptides discussed above forthe additional protein or peptide within the chimeric polypeptide.Again, sequences encoding Bt Cry proteins are preferred forincorporation into the plant. Incorporation of the additionalsequence(s) for the protein(s) or peptide(s) other than as part of thechimeric polypeptide allows for the independent expression of thechimeric polypeptide and additional protein(s) or peptide(s).

Plants suitable for transformation with the vectors of the invention maybe selected from a broad range of plants including cereal crops,vegetable, fruit and other food crops, forage crops and turf plants,fibre crops, timber and pulp and paper plants, shelter-belt plants andtree crops, ornaments and flower plants, culinary plants, medicinalplants and herbs and plants grown to produce beverages and plants grownfor smoking.

Examples of cereal crops include wheat, rice, barley, maize, oats,millet, sorghum and rye.

Examples of vegetable, fruit and other food crops include root cropssuch as potato, sweet potato, beetroot, parsnip, turnip, swede andcarrot, cucurbits such as cucumbers, pumpkins, squash, marrow,courgettes and watermelon, brassicas such as cauliflower, cabbage,oilseed rape, brussels sprouts and broccoli corn, tomato, lettuce,celery, onions, garlic, legumes such as lentils, green beans, limabeans, haricot beans, red kidney beans, kudzu beans, mung beans,broadbeans, soybeans, chickpeas, peas, and peanuts, apple, pear,kiwifruit, tamarillo, feijoa apricot, plum, citrus such as orange,lemon, tangelo, grapefruit, uglifruit and mandarin, pineapple, peach,nectarine, cherry, berries, olives and sugarcane.

Examples of forage crops and turf plants include legumes such as clover,alfalfa, lotus, trefoil and lucerne and grasses and other graminaceousplants such as ryegrass, browntop, fescue, cocksfoot, kikuyu and,paspalum, and sorghum grass.

Fibre crops include cotton, flax, kapok and hemp.

Timber, shelterbelt, conservation, pulp and paper plants and tree cropsinclude, for example, pine, eucalyptus, spruce, fir, oak, ash, birch,beech, mahogany, rosewood, ebony, maple, teak, cedar, redwood, jarrah,chestnut, walnut, macadamia nut, poplar, willow, cypress, camphor,mulberry, marram grass and rubberplant.

Ornamental shrubs, trees and flower plants include roses, petunias,orchids, carnations, chrysanthemums, daisies, tulips, lilies,gypsophylla, hibiscus, rhododendrons, conifers, camellias, hebes,lavender, lupins, tussock, ferns and native plants.

Culinary plants include herbs such as basil, rosemary, oregano, bay, andspices such as cinnamon, mace, tumeric, and sage.

Medicinal plants include poroporo, opium poppies, coca, marijuana,camomile, comfrey, foxglove and belladonna.

Plants used to produce beverages include tea, coffee, hops and cocoa.

Plants used for smoking include tobacco.

Plants transformed with the vectors of the invention direct expressionof the plant-noxious proteins in the vacuoles of the plant cells. Theprotein is effectively sequestered into the vacuole. Where the proteinis a pest control protein, when a pest feeds on the plant expressing apest control protein, the plant cell components mix together allowing asubstance to be controlled (e.g. biotin) to be bound by the bindingprotein, or alternatively degraded by enzyme (e.g. in the case ofthiamine). This essentially deprives the pest of the vitamin it requiresleading to stunted growth and death.

The effect of biotin deprivation is often manifested in the failure ofthe immature stages of the pests to complete the process of moultingfrom one developmental stage to the next as demonstrated in Examples 6to 13.

In a further aspect, the invention also provides a transgenic plantexpressing pesticidally effective concentrations of pest controlprotein.

In one preferred aspect, there is provided a plant expressinginsecticidally effective concentrations of a biotin-binding protein.Also provided are plants expressing combinations of biotin-bindingproteins and other pest control proteins as discussed above.

The present invention has application in producing plants resistant to abroad range of pests in the larval stage including moths, beetles,weevils, caterpillars, borers, budworms, armyworms, bollworms,rootworms, webworms, aphids, bugs, crickets, locusts, grasshoppers,grubs, flies, fruitflies, leafininers, plant hoppers, earwigs, scaleinsects, thrips, and springtails. Plants of the invention may also beresistant to other invertebrate pests of plants such as mites and liceand other pests and pathogens which have a vitamin requirementespecially for biotin, particularly those which undergo a moultingprocess as part of their development:

List of most Preferred Pests:

-   Order Lepidoptera:    -   cotton bollworm (Helicoverpa armigera)    -   tropical army-worm (Spodoptera litura), also S. littoralis, S.        exigua    -   European corn-borer (Ostrinia nubilalis)    -   tobacco horn worm (Manduca sexto)    -   loopers (Chrysodiexis spp.)    -   rice stem borer (Chilo suppressalis)    -   porina (Wiseana spp.)    -   cutworms (Agrotis spp.)    -   diamondback moth (Plutella xylostella)    -   potato tuber moth (Phthorimaea operculella)    -   codling moth (Cydia pomonella)    -   Indian meal moth (Plodia interpunctella)    -   gypsy moth (Lymantria dispar)-   Order Coleoptera:    -   argentine stem weevil (Listronotus bonariensisμ)    -   clover root weevil (Sitona lepidus)    -   grass-grubs (Costelytra zelandica, Odontria spp.)    -   corn rootworm (Diabrotica virgifera)    -   rice and wheat weevils (Sitophilus spp.)    -   mealworms (Tenebrio molitar)    -   flour beetles (Tribolium confusum)        Order Orthoptera:    -   black field cricket (Teleogryllus commodus)    -   locusts (Locusra migratoria)        Order Hymenoptera:    -   Sawflies (Sirex spp., Nematus olgospilus)-   Order Thysanoptera:    -   Western Flower thrips (Frankliniella occidentals)-   Order Diptera:    -   Hessian flies (Mayetiola destructor)-   Mites (Class Arachnida)-   Order Acari    -   two-spotted mite (Tetranychus urticae)    -   European red mite (Panonychus ulmi)

The applicants have also demonstrated that plants of the invention willnot cause significant mortality of desirable insects such as adulthoneybees feeding on pollen (see Example 14). Some specificity of actionis also shown where non-moulting, adult stages of insects such asweevils, and invertebrates that do not moult, such as nematodes, slugsor snails, are unlikely to be harmed by feeding on these plants. Hence,plants produced according to the invention have a broad spectrum of pestresistance for invertebrates that moult, particularly insects, as partof their development process.

In a further aspect the invention provides a method of imparting pestresistance to plants comprising transforming the plants with a vectoraccording to the present invention.

The method may also be effected by transforming isolated plant cells ortissues and generating plants from the transformed cells or tissue usingstandard culture techniques. Plants at any stage of development, partsthereof, plant cuttings, seeds, plant cells, and cell and tissuecultures transformed with vectors of the invention form further aspectsof the invention.

Transformed plants can be used in conventional breeding programmes totransfer the DNA sequences of the invention.

The plants of the invention may be grown en masse. However, it is alsofeasible to use a smaller number of plants as “bait” plants within acrop area. Only the bait plants would include the insect controlproteins. To ensure preferential targeting of bait plants by pests,attractants such as colour, hormone and scent lures may be used on oraround the bait plants.

Alternatively, bait plants may be plants which a target pest has apreference for compared with the crop being grown. For example, it hasbeen shown that rootworm have a preference for Taiuia over soybean andmaize. Such bait plants may also be used in conjunction withattractants.

In another aspect, the present invention also provides a compositioncomprising a chimeric polypeptide of the invention and a carrierdiluent, excipient or adjuvant therefor. In another composition aspect,there is provided a composition comprising plant material produced inaccordance with the invention formulated with agriculturally acceptableexcipients, carriers, diluents or adjuvants. The term “plant” as usedherein encompasses plants, plant parts such as leaves, roots andflowers, plant cuttings, seeds, tissue cultures, cell cultures and plantcells but is not limited thereto.

Preferably, the composition is a pesticidal composition comprising apesticidally effective amount of the polypeptide, or plant material andan acceptable carrier. These carriers include inert carriers such assurfactants, spreaders, stickers, mineral and organic granular carriers,stabilisers such as microencapsulation polymers or petroleum-basedsolvents.

Examples of surfactants, spreaders and stickers include C-Daxoil®,Codacide Oil®, D-C-Trate®, Supawet Oil®, Bond®, Boost® Penetrant,Citowett® and Freeway.

Examples of mineral granular spreaders include talcum powder, clay,silica, sand, limestone, gypsum, kaolin, montmorillonite, attapulgiteand diatomite.

Examples of organic granular spreaders include corncob granules, pecanshells, peanut hulls and recycled paper fibre.

Examples of stabilisers include sodium tripolyphosphate, UV-absorbers(e.g. 2,4-dihydroxy benzophenone (Uvinul M-400, UM), 4 aminobenzoic acid(PBT), fluorescent brightener-28 (FB-28)), quenchers, radicalscavengers, Hindered Amine Light Stabilizers (HALS), photostabilisers(e.g. clays, chromophores) and mineral oils.

Examples of microencapsulation polymers include cellulose acetatebutyrate (CAB), ethyl cellulose (EC22 and EC 100), low and mediummolecular weight poly(methyl methacrylate) (PMML and PMMM),poly(alpha-methylstyrene) (PMS) and starch urea formaldehyde(Starch-UF).

Examples of petroleum solvents include Aromatic 100, Aromatic 200,EXXSOL D 80, NORPAR 15, VARSOL 1, ISOPAR L, ISOPAR M, ISOPAR V andORCHEX 796.

The pesticidal composition can be applied to plants in the form ofsprays, dusts, or other formulations commonly employed in makingpesticides. In the case of the plant material containing composition thematerial will be present in a dispersable or finely divided form tofacilitate spraying onto plants to protect against pest attack. Suchsprays would be useful in reducing pest numbers, whether the bindingproteins, especially biotin-binding proteins, or degrading enzymes, hadbeen released during processing via rupturing of the vacuoles, or not.If the vacuoles remain intact, then the proteins or enzymes will bereleased as the pests feed upon the preparation, and as such theinvention may have utility as a mechanism for slow release of theseproteins or enzymes, or any other proteins directed to the vacuole bythe vector.

The compositions may further include one or more antifungal, antiviral,antimicrobial or pest control proteins all as discussed above. The useof these compositions in combination with the plants of the inventionmay be additive or synergistic, achieve broader spectrum control andreduce the risk of resistance developing.

Combinations particularly contemplated herein are compositionscomprising proteinase inhibitors or insecticidal proteins such asBacillus thuringiensis Cry proteins or biopesticides such as insectviruses or entomopathogenic fungi. Cry proteins including Cry1Ac,Cry1Cb, Cry1Da, Cry1F, Cry5 and Cry9A are preferred. The applicants havesurprisingly found that plants transformed with biotin binding proteinsand treated with Bt insecticidal protein exhibited synergistic toxiceffects on pests (see Example 18). This suggests that plants containingchimeric genes expressing both biotin binding proteins and Bt proteinswill be highly effective in protecting plants from pest attack. It islikely that such plants will be more toxic than those expressing eitherprotein singly.

In another embodiment, the compositions of the invention can be used inconjunction with transgenic plants other than those of the invention.These other transgenic plants, for example, may incorporate genesconferring fungal, viral, microbial or herbicide resistance; genesconferring early ripening, heat stability, increased accumulationability of desired products such as starch or cellulose or any otherdesirable trait as are known in the art. The composition of theinvention when applied to the transgenic plant may also achieve thedesirable results discussed above with plants of the invention.

In another embodiment, a composition of the invention may be applied toharvested material to prevent pest damage in storage. In an extrapolatedapplication, the compositions may similarly be used in plant derivedproducts such as flours, meals, cereals and the like to prevent orcontrol pest infestation.

Also provided by the present invention is a method for controlling orkilling pests comprising administering to said pest an amount of achimeric polypeptide of the invention, which includes a sequenceencoding a pest control protein, effective to control or kill said pest.

In one embodiment of the method, the chimeric polypeptide isadministered with a second pest control protein, wherein the combinationprovides more effective control than administration of the second pestcontrol protein alone. It will also be appreciated that more complexcombinations of pest control proteins including a polypeptide of theinvention are feasible. Most commonly, two to five additional pestcontrol proteins will be used. However, the methods and compositions arenot limited thereto. The additional pest control plants may comprise anyof those already discussed. A preferred additional pest control proteinis a Bt protein, especially a Cry protein.

In a related aspect, also provided is a method of controlling or killingpests, the method comprising administering to said pest plant materialof the invention which includes a sequence encoding a pest controlprotein. Compositions of the invention may also be used in these pestcontrol methods.

It will also be appreciated that a further method for controlling pestattacks on plants of the invention expressing a pest control protein,comprises treating those plants with a Bt protein or compositionincorporating same.

As discussed above, the pests against which the invention is mosteffective are the immature stages of insects, including larvae, grubs,nymphs and instars. Administration may be achieved according to anysuitable method known in the art. For example, through plant material,sprays, mulches, baits, dusts or other compositions which the pest to becontrolled takes up through feeding, inhalation, transdermal absorptionor other administrative route. Pests which may be killed or controlledusing this method include those discussed above and particularly thosereferenced in the accompanying Examples and those pests belonging to thesame insect orders as those referenced in the accompanying Examples.

It will be appreciated that the above description is provided by way ofexample only and that variations in both the materials and techniquesused which are known to those persons skilled in the art arecontemplated.

Non-limiting examples illustrating the invention will now be provided.

EXAMPLE 1 Experimental Details Concerning the Preparation of Constructs

Materials:

Custom primers were synthesized by Life Technologies. SubcloningEfficiency DH5 competent Cells were purchased from Life Technologies andthe Hybaid Recovery Plasmid Mini Prep Kit from Hybaid Limited. Allenzymes, unless otherwise stated were purchased from Promega AmpligaseThermostable DNA Ligase and Reaction Buffer and GELase were purchasedfrom Epicentre Technologies and Polymerase Chain Reaction (PCR) reagentsfrom Perkin Elmer.

The Avidin cDNA (pGEMav) carried on the plasmid pGEM13 was supplied byProfessor M. S. Kulomaa ((Department of Biological and EnvironmentalScience, University of Jyvaskyla, Finland) and the Potato ProteinaseInhibitor I (PPI-I) cDNA was isolated in this laboratory (Beuning et al.1994, GenBank Accession #L06606) and cloned into pUC19.

The Streptavidin cDNA, carried on the plasmid pET3a was supplied by TheDuPont Merck Pharmaceutical Company. The Potato Proteinase Inhibitor II(PPI-II) genomic sequence was isolated in this laboratory and clonedinto pUC19 (Murray and Christeller, 1994).

Methods:

Subcloning Efficiency DH5 competent Cells were used for general cloningand amplification of recombinant plasmids and the Hybaid RecoveryPlasmid Mini Prep Kit was used for plasmid preps. Isolation and recoveryof DNA fragments was achieved by agarose gel electrophoresis followed bytreatment of excised gel bands with GELase.

DNA Sequencing and Computer Analysis:

DNA sequencing was carried out on an Applied Biosystems (ABI) DNASequencer using dye terminator chemistry. Sequence analysis wasperformed using the Wisconsin Package Version 9.1, Genetics ComputerGroup (GCG), Madison, Wis.

EXAMPLE 2 Preparation of a Binary Vector Designed to Express a ChimericPolypeptide Comprising Avidin Mature Peptide Fused to a PotatoProteinase Inhibitor I Signal Peptide

Methods:

A one-step PCR-based mutagenesis method employing the combined use of athermostable DNA polymerase and thermostable DNA ligase (Moore andMichael, 1995), was used to prepare a construct comprising the sequenceencoding the mature Avidin polypeptide (Gope et al. 1987) fused to aPPI-I signal sequence. A Bgl II site was produced downstream of thePPI-I leader sequence at-positions 92-97 of the PPI-I coding sequenceand a BamH I site was created upstream of the sequence encoding themature Avidin polypeptide, at positions 65-70 of the sequence encodingthe Avidin protein, as shown in FIG. 1 and FIG. 2 respectively. Thesetwo restriction sites have compatible cohesive ends.

Primers:

Forward M13 (lacZ) Primer [Perkin Elmer] (SEQ ID NO:12):

-   5′-GCCAGGGTTTTCCCAGTCACGA-3′

Reverse M13 (lacZ) Primer [Perkin Elmer] (SEQ ID NO:13):

-   5′-GAGCGGATAACAATTTCACACAGG-3′

Avidin Upstream Primer (SEQ ID NO:14):

-   5′-GCACACCCGGCTGTCCACCTG-3′    Phosphorylated Mutagenic Primers

PPI-I mutagenic primer (SEQ ID NO:15):

-   5′-PGATGGACCAGAGATCTTAGAAC-3′

Avidin mutagenic primer (SEQ ID NO:16):

-   5′-PGGCTCCCGGGATCCCTGCCAG-3′    Amplification/Mutagenesis Reactions:

To generate mutant products a total PCR reaction volume of 50 μL with aneffective 1 X Ampligase Reaction Buffer [20 mM Tris-HCl (pH 8.3 at 25°C.), 25 mM KCl, 10 mM MgCl₂, 0.5 mM NAD and 0.01% Triton X-100] was usedwith the following conditions:

-   -   100 pmol each outer primer    -   1 mol phosphorylated mutagenic primer    -   40 nmol each dNTP    -   0.1 mmol dithiothreitol    -   5 U Taq DNA polymerase    -   5 U thermostable DNA ligase    -   1 ng recombinant plasmid DNA template

Reactions were first incubated at 94° C. for 3 min., followed by 30amplification cycles performed as follows:

-   -   94° C., 1 min.    -   40° C., 1 min.    -   65° C., 6 min.

Amplification cycles were followed by a final extension at 65° C. for 7min.

Restriction analysis of amplification products from both mutagenesisreactions revealed mutant product to be present, but only at a maximumof 5% of the total product. To increase the yield of mutated product,Bgl II (for PPI-I mutagenesis) and BamH I (for Avidin mutagenesis)digestion products were ligated and then used as template for a secondamplification reaction using outer primers only (Avidin Upstream andReverse M13 (lacZ) for Avidin; Forward M13 (lacZ) and Reverse M13 (lacZ)for PPI-I). For PPI-L greater than 95% of second round amplificationproduct had the desired Bgl II site and approximately of the secondround product for Avidin mutagenesis possessed the BamH I site.

The mutated PPI-I amplification product was digested with Bgl II and SalI and the mutated Avidin product with BamH I and Hind III. The PPI-Ileader sequence and the coding sequence for the Avidin mature proteinwere isolated and recovered for cloning along with Xho I/Hind IIIdigested non-recombinant pART7 vector (Gleave, 1992). These threespecies were ligated, resulting in recombinant pART 7 [refer FIG. 5] andthe sequence of the chimeric gene was checked. Subsequently, theexpression cartridge containing the gene fusion was cloned into the NotI site of pART7 vector (Gleave, 1992) and this construct [refer FIG. 7A]was mobilized to Agrobacterium tumefaciens (strain LBA4404) by standardtri-parental mating techniques (Ditta et al. 1980).

Discussion:

The resulting PPI-I/Avidin fusion protein has a total of 161 amino acidsas shown in FIG. 8. The first 31 amino acids are PPI-I sequence andsince the leader sequence comprises the first 23 amino acids, theoriginal patterning of amino acids around with the site for cleavagebetween the signal sequence and the mature protein is retained. Thereare two single base pair changes in the gene fusion sequence relative tothe predicted sequence. These changes are presumably the result of PCRerror. One change is silent and the other results in an amino acidchange from Serine to Proline at position 17 of the PPI-I signalsequence.

EXAMPLE 3 Preparation of a Binary Vector Designed to Express a ChimericPolypeptide Comprising Synthetic “Core” Streptavidin Peptide Fused to aPotato Proteinase Inhibitor II Signal Peptide

Methods:

A fused gene was prepared comprising the sequence encoding Synthetic“Core” Streptavidin (Thompson and Weber 1993) fused to a PPI-II signalsequence. The Streptavidin cDNA, carried on the plasmid pET3a was clonedinto the EcoR I/Xba I sites of pUC19 (FIG. 3). The PPI-II signalsequence (FIG. 4) which contains an intron was isolated from recombinantplasmid using PCR with a sense primer binding to pUC19 and an antisenseprimer incorporating an EcoR I site into a 5′ overhang. The primers wereas follows.

sense primer (SEQ ID NO:17):

-   5′-CTG CAG GTC GAC TCT AGA GGA-3′    antisense primer (SEQ ID NO:18):-   5′-GGT GAA TTC TTA GTA CAG ATC TTC GCA-3′    Amplification Reaction:

A total PCR reaction volume of 50 μl with an effective 1 X PCR Buffer[10 mM Tris-HCl, pH 8.3 and 50 mM KCL] was used with the followingconditions:

-   -   20 pmol each primer    -   15 nmol each dNTP    -   2.0 mM MgCl₂    -   5 U Taq DNA polymerase    -   1 ng recombinant plasmid DNA template

Reactions were first incubated at 94° C. for 2 min., followed by 30amplification cycles performed as follows:

-   -   94° C., 1 min.    -   50° C., 1 min.    -   72° C., 1 min.

Amplification cycles were followed by a final extension at 72° C. for 7min.

The PCR product representing the PPI-II signal sequence was digestedwith Sal I and EcoR I. The recombinant plasmid pUC 19/Streptavidin cDNAwas digested with EcoR I and Xba I and the Streptavidin cDNA wasisolated from the vector and recovered. Non-recombinant pUC19 wasdigested with Sal I and Xba I and the three species were ligated toproduce a construct comprising the gene fusion cloned into the Sal I andXba I sites of pUC19. The sequence of the gene fusion was checked andsubsequently cloned into the Xho I and BamH I sites of the pART7 vector[refer FIG. 6]. The pART7 expression cartridge containing the genefusion was then cloned into the Not I site of pART27 and this construct[refer FIG. 7B] was mobilized to Agrobacterium tumefaciens (strainLBA4404) by standard tri-parental mating techniques.

Discussion:

The resulting PPI-II/Streptavidin fusion protein has a total of 168amino acids as shown in FIG. 9. The first 36 amino acids are PPI-IIsequence. Five of these amino acids follow the cleavage site, preservingthe amino acid pattern around this position. The nucleotide sequence ofthe PPI-II signal sequence includes a 119 bp intron (Murray andChristeller, 1994).

EXAMPLE 4 Immunodetection of Avidin in Transgenic Tobacco

Methods:

1. Tissue Print

Samples were taken from the top 8 leaves of a tobacco plant expressingavidin (PLA2/9 #1). Four plants not expressing avidin were used ascontrols (PLA 2/3, NT12, GUS1 and JB3-13.

Pieces of transgenic tobacco leaves 1×1 cm were frozen at −20° C. for 20min, allowed to thaw and printed on to nitrocellulose using mechanicalpressure.

Labelling Protocol:

Printed nitrocellulose membranes was washed in PBS-T (phosphate bufferedsaline with 0.1% Tween 20) for 20 min, blocked in 0.1% BSA-C (Aurion)for 15 min and incubated in 1:1000 anti-avidin (Sigma A-5170) diluted inblocking buffer for 1 h (as a control for non-specific binding, thislast step was deleted in duplicate sets of prints). The membrane wasthen washed in PBS-T, incubated in goat anti-rabbit IgG-gold (10 nm)(Sigma), washed again in PBS-T, then in double distilled water anddrained. Finally the membrane was silver enhanced (BioCell silverenhancement kit) for 15 min. Enhancement was stopped by washing indistilled water.

Results:

The nitrocellulose membrane silver enhanced (turned brown) over most ofthe tissue print area in the smallest top leaf. In all other leaves thesilver enhancement was detected mainly towards the cut edges of leafmaterial. There was no silver enhancement on the prints from controlplants or on the prints made in the absence of the anti-avidin antibody.This labelling protocol also acts as a test of the labelling procedure.

2. Embedded material

Pieces 1×1×5 mm of transgenic tobacco leaf(Pla 2/9 #1)were filed in 2%paraformaldehyde and 2.5% glutaraldehyde in 0.1M phosphate buffer undervacuum for 1 h. The material was post-fixed in 1% osmium tetroxide 1 h,dehydrated in an ethanol series and embedded in Spurrs resin. Pieces ofnon-transgenic tobacco (control material) were prepared in a similarmanner. Sections were cut 300 nm thick for light microscopy (LM) andmounted on Poly-L-lysine coated slides. Sections for electron microscopy(EM) were cut 130 nm thick (gold) and mounted on carbon/formvar coatednickel grids.

Labelling Protocol:

For light microscopy the sections had a Pap pen ring drawn around themto contain the incubation liquid. The protocol for LM and EM were thesame thereafter. The sections were etched for 30 min in 10% hydrogenperoxide to remove the osmium, blocked in 0.1% BSA-c for 15 ml,incubated in anti-avidin 1:500 in PBS-T for 1 h (deleted for control)and washed in PBS-T. They were then incubated in goat anti-rabbitIgG-Alexa 488 (Molecular Probes) for 1 h. The sections were then washedthoroughly in buffer and then in double distilled water.

The methodology for labelling of sections for the electron microscope(EM) was similar to that for the light microscope (LM) except goatanti-rabbit IgG-gold (10 nm) was used instead of goat anti-rabbit IgGAlexa 488.

Sections were then viewed on a fluorescence microscope. Sections (1 μmthick) were stained methylene blue/AzureII.

Results:

Sections of Pla 2/9 #1 smallest top leaf stained for light microscopyshows darkly staining bodies mesophyll, epidermal cells, and cells ofthe glandular hairs (FIGS. 13 and 14)

Immunolabelling of LM (FIG. 14) and EM (FIGS. 15 and 16) sections showedlabelling of protein-type bodies in the vacuoles of mesophyll cells(both spongy and pallisade) and in glandular hairs (FIGS. 15 and 16).The protein bodies were usually condensed into one body which wassometimes seen as a ring. There was no labelling in the vascular tissueor in the trichomes. Control material did not label.

Conclusions:

The results indicate that avidin is synthesized in most common celltypes in tobacco leaves. The bulk of the protein appears to betransported to the vacuole and deposited as a protein body within thisorganelle.

EXAMPLE 5 ELISA Assay of Avidin and Streptavidin

The following general ELISA assay technique was used for assaying foravidin and streptavidin where indicated in the following examples.

Method:

-   1. Plant material was ground with 2 volumes (w/v) of ice cold 0.05    μM sodium phosphate (pH 7.5) containing 5% polyvinylpolypyrrolidone.    This was centrifuged and the supernatant used for analysis. In order    to construct standard curves control plant material was ground in    the above buffer with and without 0.2 mg/mL avidin or streptavidin    and centrifuged.-   2. Generally 10 μL of extract and 90 μL of coating buffer (15 mM    sodium carbonate, 46 mM sodium bicarbonate, pH 9.6) were mixed in a    96 well microtitre plate and incubated at 4C overnight. Each sample    was duplicated and standards consisted of various proportion of    control plant extract/added protein extract to the same total    extract volume as the samples.-   3. Plates were washed (3×) in phosphate-buffered saline (PBS)    containing 0.02% Tween 20 (PBST) and incubated for 1 hr in 100 μL of    PBST containing 0.5% gelatin.-   4. Plates were washed (3×) in PBST and incubated for 1 hr in 100 μL    of PBS containing either polyclonal rabbit anti-avidin or    anti-streptavidin antibodies.-   5. Plates were washed (3×) in PBST and incubated for 1 hr in 100 μL    of PBS containing goat anti-rabbit antibody linked to alkaline    phosphatase.-   6. Plates were washed (3×) in PBST and, after addition of 100 μL of    0.1 M diethanolamine (pH 9.8) containing 0.5 mM MgCl₂ and 0.5 mg/mL    p-nitrophenyl phosphate, assayed at 410 nm in a microtitre plate    reader. Initial rates of samples were determined by linear    regression over 5-10 mins and compared to rates obtained for the    duplicate standard curves (8 avidin or streptavidin concentrations)    on each microtitre plate.-   7. Concentrations of avidin and streptavidin in the samples were    determined as the mean molar concentration in the tissue assuming    that the specific gravity of plant tissue is one and molecular    weights for avidin and streptavidin of 15600 and 16473 (for the    standard) respectively.

EXAMPLE 6 Toxicity of Whole Tobacco (Nicotiana Tabacum) PlantsExpressing Avidin to Potato Tuber Moth Larvae (Phthorimaea operculella)(Lepidoptera: Gelechiidae)

Constructs:

-   Non-transformed control plants    -   2 plants (NT 1, NT 2)-   Control plants transformed with pumpkin fruit chymotrypsin inhibitor    (PFCI) but not expressing the protein    -   3 plants (JB3/1, JB3/2, JB5/1)-   Tobacco plants transformed with the avidin gene with a PPI-I    targeting sequence (Example 2 above)    -   6 lines (PLA2/2, PLA2/7, PLA2/9, PLA2/13, PLA2/20, PLA2/24), 4        clonal plants per line        Trial Design:        Trial 1:

The tobacco plants were removed from tissue culture and potted infertilised potting mix (Smiths® general potting mix) before being placedin large ventilated acetate containers (220×300 mm) in a containmentglasshouse unit at 22±5° C. They were watered daily to maintain highhumidity and soil moisture content.

Eight days later, when plants were well established with at least fourto five small leaves, ten neonate potato tuber moth (PTM) larvae wereplaced on each tobacco plant, usually two per leaf. Prior to inoculationthe larvae were weighed in batches of five (since single larvae are toosmall to give an accurate reading). TM larvae were obtained from alaboratory culture reared on potato tubers following the same basicprocedure as Broodryk (1971) and Meisner et al. (1974).

Trial 2:

One week after Trial 1 was completed, the tobacco plants were cut backto the second node and allowed to regenerate leaves. When the plants haddeveloped four to five leaves (in approximately 11 days) they were eachinoculated again with ten neonate PTM larvae, usually two per/leaf,weighed in batches of five prior to inoculation as above.

Trails 1 and 2:

Inoculated plants were kept individually in acetate containers in thecontainment glasshouse unit at 22±5° C. for nine days. Under theseconditions growth of control larvae is exponential from hatch to ninedays, but after this growth rate slows as pupation approaches. Hence inorder to compare growth rates of larvae on control and transgenicplants, the trial was concluded after nine days. Damaged leavescontaining larvae were removed, and larvae were dissected out of theirmines within the leaf or stem tissue. The intention was to weigh thelarvae at this point in order to estimate growth rates, but, except forthose on control plants, larvae were mostly dead, dried and shrivelled.Consequently head capsules were measured so that the instar reached atdeath could be recorded.

Level of Expression of the Avidin Protein

Results:

The level of expression of avidin in each of the plant lines wasquantitated using chemilumenessance detection of avidin protein fromwestern blots of leaf tissue, compared to authentic avidin standards andexpressed as percentage of total leaf protein. These levels are given inTable 1 below.

TABLE 1 The level of expression of avidin as % of total leaf protein,determined using the chemiluminescence method Avidin expression PlantLine % total leaf protein (μM)* PLA2/2 0.07 (0.90) PLA2/7 0.10 (1.23)PLA2/9 0.07 (0.90) PLA2/13 0.06 (0.77) PLA2/20  0.065 (0.83) PLA2/240.06 (0.77) *The chemiluminescence method was used to estimate avidinexpression as % total soluble leaf protein. In later Examples, an ELISAmethod (Example 5) was used to estimate the expression levels as μM.Hence these values were converted to μM. Avidin expression was measuredin clones of these original plants using the ELISA method and resultsgiven in Example 8, Table 5. These levels are about three times higherthan those given in Table 1. This may reflect the fact that, in these #trials the measurements were done on plants still in tissue culturewhereas those in Example 8 wree done on large leaves from mature plants.Mortality of PTM Larvae Feeding on Whole Tobacco Plants Expressing theAvidin Gene.Trial 1:

Good recovery rates of larvae from both control and transgenic plantswere obtained: 86% from controls and 76.7% from transformed plants. FIG.10 clearly shows the high mortality PTM larvae after feeding for ninedays on whole transgenic tobacco plants expressing the avidin genecompared to both non-transformed control plants and control plantstransformed with, but not expressing, the pumpkin fruit chymotrypsininhibitor (PFCI) gene.

The majority of dead larvae were recovered from mines where they haddied at the “cutting face”. A few (5% of dead larvae) were recoveredfrom the surface of leaves, having generally left a mine close by. It ismost likely that the majority of larvae not recovered had died in thisway and had fallen off the leaves. Some mines were found withoutoccupants. However, there was no evidence that larvae had started andabandoned mines on several occasions as we have previously observed inanother experiment in which larvae were fed on tobacco expressing cry1Ac and cry 9Aa2 genes (Gleave et al. 1998).

PTM larvae undergo four instars during their development. In order todefine the stage of development of the larvae at death, head capsulewidths were measured using a micrometer eye-piece. All control larvaewere alive and most were third instars. None of the larvae recovered onany of the plants expressing avidin had reached third instar beforedeath and many had died during or just after the moult from first tosecond instar, as evidenced by the fact that the ecdysed cuticle wasstill attached. This reflects results in earlier trials with avidinincorporated into diet. Table 2 below gives a breakdown of instars oneach plant line.

TABLE 2 Number of larvae at each instar recovered from transgenictobacco plants expressing avidin in Trial 1 Neonates Number of larvae atPlant line inoculated 1st instar 2nd instar 3rd instar 4th instar NTcontrol 20 0 1 18 0 JB control 30 0 0 23 1 PLA2/2 40 3 28 0 0 PLA2/7 404 23 0 0 PLA2/9 40 2 27 0 0 PLA2/13 40 1 25 0 0 PLA2/20 40 2 27 0 0PLA2/24 40 4 25 0 0Trial 2:

Again there were good recovery rates of larvae from both control andtransgenic plants: 88% from controls and 88.8% from transformed plants.FIG. 11 reflects the results of the first trial showing high mortalityof PTM larvae fed on whole trans genic tobacco plants expressing theavidin gene compared to those on control plants. In fact a total of onlyfour live larvae were recovered from all avidin expressing plants (<1.7%survival), whereas only three larvae had died on the control plants (94%survival).

Head capsule widths of larvae were measured and the number of recoveredlarvae at each instar is given in Table 3. As in the first trial, noneof the larvae recovered from any of the plants expressing avidin hadreached third instar before death and many had died during or just afterthe moult from first to second instar again the ecdysed cuticle wasstill attached in many cases.

TABLE 3 Number of larvae at each instar recovered from transgenictobacco plants expressing avidin in Trial 2 Neonates Number of larvae atPlant line inoculated 1st instar 2nd instar 3rd instar 4th instar NTcontrol 20 0 3 12 0 JB control 30 0 1 24 3 PLA2/2 40 34 5 0 0 PLA2/7 4025 9 0 0 PLA2/9 40 30 3 0 0 PLA2/13 40 26 11 0 0 PLA2/20 40 25 5 0 0PLA2/24 40 30 3 0 0Conclusion:

Total mortality of PTM larvae fed on tobacco plants expressing theavidin gene would have occurred if the trials had been continued beyondnine days; larvae that survived for nine days were small, shrivelled andclose to death as evidenced by their minimal response when touched by afine sable paint brush.

Avidin expressed in tobacco plants is highly toxic to PTM larvae and hasdefinite potential in the development of pest resistant crop cultivars.

EXAMPLE 7 Toxicity of Whole Tobacco (Nicotiana tabacum) PlantsExpressing Streptavidin to Potato Tuber Moth Larvae (Phthorimaeaoperculella) (Lepidoptera: Gelechiidae)

Constructs:

-   Non-transformed Control Plants    -   6 plants (NT21-26)-   Plants transformed with and expressing the streptavidin gene with a    PPI-II targeting sequence (Example 3 above)-(Sav)    -   6 plant lines, 5 clones per line (5, 9, 10, 14, 23, 26)    -   2 plant lines, 3 clones per line (25, 28).        Trial Design:        Trial 1:

The transformed tobacco plants were removed from tissue culture, plantedin fertilised potting mix (Smiths® general potting mix) and placedindividually in large ventilated acetate containers (220×300 nm) in acontainment glasshouse unit at 24±7° C. They were watered regularly tomaintain high humidity and soil moisture content until well established.

Eleven days later, five neonate potato tuber moth (PTM) larvae wereplaced on each tobacco plant. Prior to inoculation the larvae wereweighed in batches of five (since single larvae are too small to give anaccurate reading on a 5-place balance). PTM larvae were obtained from alaboratory culture reared on potato tubers following the same basicprocedure as Broodryk (1971) and Meisner et al. (1974).

Trial 2:

On completion of Trial 1, the tobacco plants were cut back to the secondnode and allowed to regenerate new leaves. When the plants had developedat least four to five leaves they were each inoculated again with fiveneonate PTM larvae as above. Unfortunately some individual plants diedduring this process and so fewer clones were tested for some lines inthe second trial.

Trials 1 and 2:

Inoculated plants were kept individually in acetate containers in thecontainment glasshouse unit at 24±7° C. for nine days. Under theseconditions growth of control larvae is exponential from hatch to ninedays, but after this growth rate slows as pupation approaches. Hence inorder to compare growth rates of larvae on control and transgenicplants, the trial was concluded after nine days. Damaged leavescontaining larvae were removed, and larvae were dissected out of theirmines within the leaf or stem tissue. The intention was to weigh thelarvae at this point in order to estimate growth rates, but, except forthose on control plants, the larvae were mostly dead, dried andshrivelled. Consequently head capsule width was measured for all larvaeretrieved so that the instar reached at death could be recorded.

Results:

Level of Expression of the Streptavidin Protein:

The level of expression of streptavidin in each of the plant lines wasmeasured using the technique described in Example 5. These levels aregiven in Table 4 below.

TABLE 4 Expression of streptavidin in tobacco plants Plant LineExpression of Streptavidin (Savα) μM (s.e.)  5 12.802 (0.834)  9 17.818(0.059) 10 11.404 (0.896) 14 18.178 (0.560) 23 24.524 (0.042) 25 21.703(0.842) 26 16.306 (1.831) 28 15.788 (0.260)Mortality of PTM Larvae Feeding on Whole Tobacco Plants Expressing theStreptavidin Gene:Trials 1 and 2:

Recovery of larvae was good from control plants in both trials (88.6 and92% respectively) and from transgenic plants (78.3 and 83% respectively)and similar to that reported in the trials with tobacco expressing theavidin gene (Example 6). FIG. 17 shows the number of live and deadlarvae recovered nine days after inoculation, from each plant line inboth trials. In Trial 2 there was total mortality on all plant lines,but in Trial 1 there were a few survivors after nine days on some plantlines: of the 25 larvae initially placed on the plants, two survived online 5 and one each on lines 9, 10, 23 and 28. However, all of these“survivors” were close to death. In contrast, there was no larvalmortality on control plants in either trial. As in Example 6, themajority of larvae had died within the mines in the leaves and only afew dead larvae were found on the leaf surface after abandoning theirmines.

Head capsule widths of all larvae were measured after they were removedfrom their leaf mines to determine their stage of development. Larvaerecovered from non-transgenic (NT) plants were all alive in both trialsand all but two had reached 3rd or 4^(th) instar. In contrast, themajority of larvae feeding on the transgenic plants had died at 1^(st)or 2^(nd) instar (FIG. 18). Most of these had died just prior to orduring the ecdysis from 1^(st) to 2^(nd) instar as was evidenced by thenumber of dead larvae with ecdysed skins and head capsules stillattached.

Conclusion

Tobacco plants expressing the streptavidin gene were highly insecticidalto potato tuber moth larvae. Larval mortality occurred on all plantstested expressing the gene and the majority of larvae died just priorto, during, or immediately after ecdysis between the 1^(st) and 2^(nd)instar.

EXAMPLE 8 Toxicity of Avidin Expressed in Tobacco (Nicotiana tabacum)Leaves to Larvae of the Common Cutworm Spodoptera litura (Lepidoptera:Nictuidae) and the Cotton Bollworm (Tomato Fruitworm, Cornear Worm)Helicoverpa armigera (Lepidoptera: Noctuidae)

Constructs:

Control lines:

-   Non-transformed control plants:    -   4 plants (NT11, NT12, NT13, NT14)-   Control plants transformed with PRD400 vector with pumpkin fruit    chymotrypsin inhibitor (PFCI) gene but which do not express the    transgene:    -   8 plants (6 independent transformants) (JB3-1C/AB. JB3-1,        JB3-13, JB3-15, 2 clonal JB3-16 plants, and 2 clonal JB3-25        plants)        Control Plants Transformed with the pART27 Vector:    -   7 plants (all independent transformants) (art27c #1, art27c #3,        art27c #4, art27c #5, art27c #6, art27c #7, art27c #8)        Control Plants Containing the pART27 Vector with the GUS Gene        (uid):    -   4 plants (all independent transformants) (GUS 1, GUS2, GUS5,        GUS8)        Avidin-expressing Lines:    -   Tobacco plants transformed with the avidin gene with a PPI-I        targeting sequence (Example 2 above):    -   6 plant lines derived from 6 separate transformation events        (PLA2/2, PLA2/7 PLA2/9, PLA2/13, PLA2/20, PLA2/24), 4 clonal        plants per line.        Insects:

Spodoptera litura were obtained from a laboratory colony originallyestablished from moths field-collected in Queensland, Australia, whileHelicoverpa armigera were from a laboratory colony established frommoths collected in Christchurch, New Zealand. Both colonies were rearedon artificial diet as described in McManus and Burgess (1995).

Neonate S. litura larvae were placed on tobacco leaves within 12 h ofemergence from eggs. Initial larval weight was estimated from the meanweight of three samples of 100 larvae.

Neonate H. armigera larvae were placed on artificial diet for 48 hfollowing emergence from eggs, and then placed on tobacco leaves as latefirst instar larvae. Initial larval weight was determined as the mean ofthe individual weights of a randomly chosen sample of 48 larvae weighedat the beginning of the experiment.

Trial Design:

On each plant used in the experiment, Leaf 1 was designated as theuppermost (youngest) leaf which was 15 cm or more in length from leaftip to leaf base (the point at which the leaf joined the petiole). Theleaves below Leaf 1 were assigned numbers consecutively down the plantLeaves 1 and 2 were used for H. armigera as previous experiments hadshown that larvae grow best on these leaves. For similar reasons. S.litura were given Leaves 4 and 5 of the same plants.

To ensure leaves remained turgid during larval feeding each leaf was cutfrom the plant close to the stem leaving a long petiole, and eachpetiole was immediately plunged into about 20 μL of a setting solutionof 0.4% agar in a 30 mL coulter cup.

At the start of the experiment, larvae were placed on leaves from oneplant from each of the six clonal avidin-expressing lines, and on sixcontrol plants. Twelve H. armigera larvae were placed on the undersidesof Leaves 1 and 2, i.e 12 larvae×2 leaves×6 plant lines=144 larvae, andan equivalent number of control larvae were used. For S. litura; 15larvae were placed on the upper surfaces of Leaves 4 and 5, i.e. 15larvae×2 leaves×6 plant lines=180 larvae on both avidin and controltreatments.

Each leaf with larvae was placed in a 300×210×80 mm plastic storage boxlined with paper towels and with a snap-on lid. Larvae and leaves werechecked daily, and leaves were replaced with new ones from fresh plantsas necessary so that larvae could feed ad libitum. Throughout theexperiment, larvae on avidin plants were fed leaves from within the sameclonal line (e.g. PLA2/2 or PLA2/7), and larvae on control plants werekept on the same genetic plant type (NT or JB-3 or art27c or GUS). Whennecessary, leaves of the equivalent physiological age from previouslyused plants were utilised.

The experiment was conducted in a controlled temperature room at 24.5±1°C. and 60% relative humidity, with a 16:8 h light:dark cycle.

Larvae were first weighed and survivors counted at Day 8, and then atregular intervals throughout the experiment until death or untilpupation had begun in a treatment.

Determination of Avidin Expression Levels:

To measure expression levels in plants fed to larvae, two leaf samplesof 8 cm² were taken from Leaf 4 of all avidin plants used in the trial.One sample was taken just before larvae were initially placed on theleaf, and the other a few days later, following the transfer of larvaefrom the leaf onto a fresh leaf. Expression was measured as described inExample 5.

TABLE 5 Expression levels of avidin in plants Mean expression level ofavidin Number of Plant line (μM) Standard error samples PLA2/2 3.10 0.428 PLA2/7 3.29 0.23 8 PLA2/9 4.37 0.51 8 PLA2/13 3.40 0.38 8 PLA2/20 4.590.31 8 PLA2/24 4.10 0.21 8 NT 0 — 8Results:S. litura

As the same pattern of response was observed in larvae on all controllines, results from the different lines were pooled. The sameobservation was made for larvae on all avidin lines, so results fromthese lines were also pooled.

Larvae on avidin-expressing plants were significantly smaller thancontrols at the first weighing on Day 8 (control plants: N=153, meanweight=0.0304 g, s.e.=0.0014; avidin plants: N=160, mean weight=0.0151g, s.e.=0.0007; P<0.001) (FIG. 19), (ANOVA, Payne et al., 1993),butthere were no differences in survival at that time. By Day 12, larvaeeating avidin plants had begun to die (P<0.001) (FIG. 20), and therewere clear differences in mean weight and total live biomass present onthe two treatments (P<0.001) (FIG. 21). By Day 15, these differenceswere even more pronounced, and after this time control larvae hadpupated, so no further control measurements were taken. Comparativelarval sizes on control and avidin plants are shown on Day 15 in FIG.19B. Differences in size and plant damage on Day 15 are shown in FIG.19C. Larvae on avidin plants steadily diminished in numbers and totalbiomass, and by day 25 all had died.

We observed that larvae feeding on avidin plants were unable tosuccessfully complete the process of moulting from one instar to thenext. Larvae on these plants appeared to stop feeding during ecdysis,and to then turn black and die while still attached to a partially shedlarval skin.

H. armigera

As with S. litura, larval responses on all control lines were the same,and results were thus pooled, as were responses on all avidin lines. H.armigera larvae fed avidin-expressing plants were smaller than those fedcontrol plants by Day 8 (control plants: N=130, mean weight=0.0909 g,s.e.=0.0031; avidin plants: N=130, mean weight=0.0375 s.e.=0.0013;P<0.001) (FIG. 22). Three days later, control larvae had continued togrow well while avidin-fed larvae had begun to die (P<0.001) (FIG. 23),and differences in biomass between the two treatments were extreme(P<0.001) (FIG. 24). No further control measurements were made after Day11 as larvae had begun to pupate. Comparative larval sizes on controland avidin are shown on Day 14 in FIG. 22B. Differences in size andplant damage on Day 14 are shown in FIG. 22C. By Day 22, all larvae onavidin plants had died.

As with S. litura, H. armigera larvae on avidin plants often died duringthe moulting process.

Conclusions:

The expression of avidin in six different transgenic lines of tobaccowas fatal to larval S. litura and H. armigera. Larvae of both theselepidopteran pest species grew rapidly and pupated on a range of nontransgenic and transgenic tobacco lines which did not express avidin.Larvae fed avidin-expressing plants were unable to develop normally orattain significant biomass, often dying during early instar moults.

These results provide further evidence of the effectiveness of theavidin construct described in this patent in protecting the plant inwhich it is expressed from insect damage.

EXAMPLE 9 Toxicity of Avidin Expressed at a Range of ConcentrationLevels in Tobacco (Nicotiana tabacum) Leaves to Larvae of the CottonBoll Worm (Tomato Fruitworm, Cornear Worm) Helicoverpa armigera(Lepidoptera: Noctuidae)

Constructs:

Control Lines:

Non-transformed Control Plants:

-   -   48 plants (NT 101-NT 148).

These were grown from seeds produced by selfed NT plants 11-14 whichwere used in the trial described in Example 8.

Avidin-expressing Lines:

Tobacco plants (T₁) were grown from seeds collected from 3 selfed plantsfrom the clonal lines PLA2/7, PLA2/9 and PLA2/13. These T₀ parentalplants had been transformed with the avidin gene with a PPI-I targetingsequence (Example 2 above), and were used in the trial described inExample 8. Twenty four plants from each of these three T₁ seed lineswere grown for the experiment and 25 of these 72 plants were selectedfor use depending on their level of avidin expression.

Insects:

Neonate H. armigera larvae from our laboratory colony (see Example 8)were placed on the leguminous host plant Lotus corniculatus and kept at18° C. for 34 days prior to the experiment. Late first instar larvaewere then transferred to control and avidin-expressing tobacco leaves.Initial larval weight was determined as the mean of the individualweights of a randomly selected sample of 48 larvae weighed at thebeginning of the experiment.

Trial Design:

To measure the effect of avidin expression level on the growth, survivaland biomass of H. armigera larvae, the 72 T₁ avidin plants describedabove were tested for expression level using the ELISA method (Example5). A leaf sample of approximately 50-60 cm² was removed from the tip ofLeaf 4 of each plant for this process. All plants were ranked accordingto their expression level, and divided into six groups representing sixnon-overlapping ranges of expression level, from “high” to “low”. Thesesix groups of plants were assigned as six treatments with different meanavidin concentrations (Table 6).

At the start of the trial, the highest expressing plant from eachtreatment group and two control plants of similar physiological formwere selected. Leaf numbers were assigned on each plant as described inExample 8, and Leaves 1 and 2 cut from each plant for use in the trial.Petioles were again immersed inserting in agar to maintain leaffreshness, and 12 H. armigera larvae were placed on the underside ofeach leaf.

As in Example 8, leaves were stored in plastic boxes, and the experimentconducted at 24.5±1° C. and 60% relative humidity, with a 16:8 hlight:dark cycle.

Larvae were weighed on Days 8, 11, 13, 14 and 15, and surviving larvaewere transferred to fresh leaves 1 and 2 from the next highestexpressing plant in each treatment croup on Days 6, 8, 11 and 16. Toensure that larvae could feed ad libitum, additional leaves were cutfrom positions immediately above or below Leaves 1 and 2 on the sameplants, and provided to larvae if necessary. Control larvae requiredmany more leaf additions than all other treatments, and thus were givenadditional leaves from a range of control plants and leaf positions.

TABLE 6 Expression levels of avidin in treatment groups of plants usedin trial Mean expression level of avidin Number of Treatment (μM)Standard error plants used 1 17.25 0.44 5 2 14.18 0.09 4 3 10.85 0.07 44 8.71 0.12 4 5 6.40 0.12 4 6 3.69 0.11 5 Control 0 — 48Results:

As there were no significant differences between larval growth, survivaland biomass on the two control treatments, the results of these twotreatments were combined.

By the time larvae were first weighed on Day 8 of the experiment,control larvae had grown larger than those in all other treatments (FIG.25) (P<0.05-P<0.0001) (ANOVA., Payne et al, 1993). These differencesincreased with time.

Comparison of larval survival curves using a log-rank test (Kalbfleischand Prentice, 1980) showed that survival on all avidin-expressing lineswas significantly reduced in comparison with control survival (P<0.001).There were no significant differences between survival on any of the sixlines expressing avidin at different levels (P=0.328).

All the larvae fed plants expressing avidin at 17.25-6.40 M failed toachieve substantial growth, and died, often during moulting, withoutpupating (FIG. 26). Two of the 24 larvae on the lowest expressing avidintreatment pupated, although they were smaller than control larvae. Oneof these pupae emerged as a moth. On the two control treatments, 31 of48 larvae successfully pupated and 19 of these emerged as moths. Thenumber of larvae successfully pupating in the control treatments wasreduced by cannibalism of prepupae by voracious late instar larvae. Thiseffect may also have reduced the rate of emergence of moths from pupaein the controls. No such effect occurred in the avidin treatmentsbecause of the extremely high larval death rate caused by the ingestionof avidin-expressing leaf material.

Accumulation of biomass on the avidin-expressing lines was negligiblecompared to that on the control lines (Fic. 27).

Conclusions:

Tobacco plants expressing avidin at levels ranging from 6.40 to 17.25 μMcaused total mortaliry of H. armigera larvae in this trial. Expressionlevels of 3.69 μM resulted in a very high level of larval mortality(92%). All plants expressing avidin at any level were protected frominsect attack as evidenced by the extremely low biomass of insects onthose plants.

EXAMPLE 10 Toxicity of Avidin and Streptavidin Incorporated intoArtificial Diets to the Pine Shoot Tip Moth, Rhyacionia Buoliana(Lepidoptera: Tortricidae)

Insects:

A laboratory colony of Rhyacionia buoliana was established by fieldcollection of late instar larvae and pupae from pine (Pinus radiata)plantations throughout Chile. Field-collected individuals which becameadults were confined in laboratory cages to allow mating. Eggs laid byadult females were collected, and larvae which emerged from these wereused in this trial.

Methods:

The avidin used in this experiment was a Calbiochem® product purchasedfrom Calbiochem-Novabiochem Corporation, La Jolla, Calif. 92039. It waslyophilized avidin from egg white, Product Number 189725, Lot Number276992.

The streptavidin was also obtained from Calbiochem-NovabiochemCorporation, and was a lyophilized solid, Product Number 189730, LotNumber B19870.

Avidin and streptavidin were incorporated into artificial diet at thefollowing concentrations in eight treatments:

-   1. control, 0 μg/mL-   2. control, 0 μg/mL-   3. avidin, 50 μg/mL-   4. avidin, 100 μg/mL-   5. avidin. 1000 μg/mL-   6. streptavidin. 50 μg/mL-   7. streptavidin, 100 μg/mL-   8. streptavidin. 1000 μg/mL

These levels are equivalent to plant expression of 3.2, 6.4 and 64 μM ofavidin, and 3.0, 6.1 and 60.6 μM of streptavidin. We have shown avidinexpression levels in tobacco ranging from 3-25 μM (Examples 8, 9 and18), and streptavidin levels of 11-24 μM (Example 7).

The artificial diet used in this experiment was a general purpose insectrearing diet based on the recipe of Singh (1983). The avidin andstreptavidin were added in aqueous solution into freshly made diet,which had cooled to 60° C.

The experiment was run in a randomised complete block design, in threeblocks, which were set up on consecutive days. Both avidin andstreptavidin, at each of the three doses, were fed to a total of 90larvae, and 180 larvae were given control diet:

-   i.e. 2 proteins×3 concentrations×30 larvae×3 blocks=540 larvae+2    controls×30 larvae×3 blocks=180 larvae

Within 12 h of hatching from eggs, neonate larvae were placed in pottlescontaining BIO-SERV® pine tip moth diet (the diet on which the colonywas reared).

At the beginning of the experiment, healthy 24 h-old larvae which hadestablished well on this diet were then transferred to 1.5 mL Eppendorftubes containing a 0.25 ml block of treatment diet, where they wereconfined individually. Initial mean larval weight was determined byweighing 100 of these healthy larvae selected for the experiment enmasse.

Larval survival was checked every seven days for the duration of theexperiment. After 14 days, larvae were weighed and transferred to newtubes with 1 μL of fresh diet. After 35 days, surviving larvae wereweighed again, and the experiment terminated.

This experiment was conducted in a temperature-controlled incubator setat 20° C., in which lights periodically switched on when the temperaturedropped below the target.

Results:

As there were no significant differences between data collected for thethree blocks of any given treatment, results for the three blocks werepooled within all treatments.

Both avidin and streptavidin at all 3 concentrations had causedsignificant reductions in larval growth by Day 14 (FIG. 28) (P<0.0001)(ANOVA, Payne et al., 1993), and these differences increased by Day 35.Both proteins were toxic to larvae, and most individuals feeding on anavidin or a streptavidin diet were dead before the end of the experiment(FIG. 29). Many of the dead larvae had died during the process ofmoulting from one instar to the next. Larvae that survived feeding ondiet containing either protein at any of the three concentrations wereclose to death. Comparison of survival curves using log-rank testsshowed all treatments reduced larval survival compared with controls(P<0.001). The highest dose of streptavidin killed larvae faster thanany of the other treatments (P<0.001), but there were no otherdifferences among the survival responses to other doses of eitherprotein. Because both avidin and streptavidin killed most larvae andprevented growth in survivors, there were very large differences betweeninsect biomass on controls and all other treatments (FIG. 30).

Conclusions:

This trial has demonstrated the high level of toxicity of both avidinand streptavidin to the pine shoot tip moth, Rhyacionia buoliana. Theseresults suggest that either of these proteins would control the pest ifexpressed in P. radiata or other host trees at levels equivalent tothose we have demonstrated for avidin and streptavidin in tobacco plants(Examples 7, 8, 9 and 18).

EXAMPLE 11 Toxicity of Avidin-painted Willow (Salix fragilis) Leaves toNeonate Willow Sawfly Larvae (Nematus oligospilus) (Hymenoptera:Tenthredinidae).

Insects:

Willow sawfly larvae (Nematus oligospilus) which had hatched within theprevious 24-hour period, were obtained from a laboratory colony rearedon small potted willow plants (Salix fragilis).

Leaf Material:

Leaves were obtained from potted willow plants (S. fragilis) grown in ashade house, the same source as those on which the larvae were reared.

Methods:

The avidin used in this trial was a Calbiochem® product, purchased fromCalbiochem-Novabiochem Corporation, La Jolla, Calif. 92039. It waslyophilized avidin from egg white, Lot 276992.

Willow leaves were weighed and a mean leaf weight obtained (194.5±13.1mg). Using this weight the amount of avidin to apply per leaf wascalculated as 65 and 130 μM delivered as 200 and 400 μg avidin/leaf.

To ensure avidin was well distributed over the leaf surfaces it wasdissolved in a 0.1% solution of the “wetter and sticker”, BondXtra®(i.e. 50 μL in 50 mL). 100 μL/leaf gave good coverage.

Solutions were painted on to leaves using a sable brush (Cirrus 110®).The brush was weighed before and after applying the solutions to theleaves and was found to absorb about one-tenth of the volume. Hence 55μL of each solution was pipetted on and applied to each side of eachwillow leaf. Leaves were allowed to air dry.

Trial Design:

Excised leaves were trimmed to fit across a Petri dish, one leaf perdish. The leaf petiole was placed in a small tube of water and paintedwith the appropriate solution. After being air-dried, the leaf was thenpushed through a hole in the side of the Petri dish. Water was topped upevery 2 days. Close cell foam supported the petiole and filled the spacearound the hole preventing the larvae escaping. The Petri dish with tubeattached was stuck to a backing board with Blu-tack® and held firmly inplace with a rubber band. The whole set up was then set vertically on aslotted board. Each Petri dish contained one willow leaf and one larvaand each treatment tested 20 larvae. There were four treatments:

-   1. controls in which leaves were untreated,-   2. 0.1% BondXtra®,-   3. 65 μM avidin in 0.1% BondXtra®,-   4. 130 μM avidin in 0.1% BondXtra®.

Larvae were weighed individually and one placed in each Petri dishcontaining a single willow leaf. Surviving larvae were weighed againafter 7, 14 and 21 days and leaves were changed after 10 and 15 days.

Results:

FIG. 31 shows survival of sawfly larvae over the first 21 days by whichtime the majority of survivors had pupated. Whilst no controls died andonly one death was recorded amongst larvae treated with BondXtra®survival of larvae on leaves coated with avidin declined steadily. Theproportion of sawfly larvae surviving to 21 days on leaves coated with65 μM avidin was 0.4, and 130 μM avidin only 0.1. Further, weight gainover the first 14 days was significantly reduced at both avidinconcentrations when compared to control larvae and those feeding onleaves treated with BondXtra® alone (FIG. 32).

At pupation, sawfly larvae form a fibrous pupal case or cocoon. At thelower avidin concentration only one out of the six larvae that reachedpupation and developed a cocoon failed to emerge as an adult. At thehigher avidin concentration, only one larvae attempted and failed topupate; no adults emerged from this treatment (FIGS. 33 and 34). In bothcases where the larva failed to emerge as an adult the fibrous pupalcase contained a shrivelled dead larval body and so ecdysis (moult) hadnot been completed.

Conclusions:

Avidin is highly insecticidal to willow sawfly larvae and, as has beenobserved in bioassays with this protein on other insect species (seeother examples), it appears to have acted both as a growth inhibitor andas a moulting inhibitor.

EXAMPLE 12 Toxicity of Avidin-Painted Lettuce (Latuca sativa) Leaves tothe Black Field Cricket, Teleogryllus commodus (Orthoptera: Gryllidae)

Insects:

Crickets were obtained from a laboratory colony of Teleogryllus commodusoriginally field collected in Northland, New Zealand. Four day oldnymphs were used in this trial. These individuals had been fed sinceeclosion from eggs on the normal colony diet for young nymphs of rolledoats, dried lucerne (Medicago sativa) meal and dog biscuits (Pedigree®PAL Meaty-Bites®).

Methods:

The avidin used in this trial was a Calbiochem® product, purchased fromCalbiochem-Novabiochem Corporation. La Jolla, Calif. 92039. It waslyophilized avidin from egg white, Lot 276992.

Green distal portions of leaves from organically grown lettuce leaf werecut into sections approximately 4×4 cm. These were painted on both sideswith three different solutions, providing three treatments:

-   1. Control solution of 0.1% (v:v) BondXtra®, a wetting, spreading    and sticking agent-   2. 4.8 μM avidin (75 μg/g fresh weight of lettuce leaf) in 0.1%    (v:v) BondXtra®-   3. 19.2 μM avidin (300 μg/g fresh weight of lettuce leaf) in 0.1%    (v:v) BondXtra®

Cricket nymphs were weighed and placed individually in 75 mL specimenpottles with ventilation holes punched in their lids, and with a 42.5 mmfilter paper disc placed in the bottom of each pottle to absorb excessmoisture. Food was replaced as necessary so that crickets could feed adlibitum on fresh leaf material. Each cricket was weighed weekly untilall individuals feeding on the avidin-painted leaves had died.

Results:

Crickets grew well on control leaves but poorly on leaves painted withavidin at both concentrations (FIG. 35). By Day 21 control crickets weresignificantly larger than those surviving avidin treatment (P<0.05)(ANOVA, Payne et al. 1993). By this time, all those on 4.8 μM avidinleaves were dead (FIG. 36) and there were few survivors on the 19.2 μMavidin treatment. By Day 35, all crickets on the 19.2 μM treatment hadalso died. Many of the avidin-fed crickets died while moulting from onenymphal stage to the next. Cricket biomass in the control treatmentsteadily increased throughout the experiment, while biomass had reachedzero in the 4.8 μM avidin treatment by Day 21, and dropped below thestarting value in the 19.2 μM treatment by this time (FIG. 37). Biomassin the 19.2 μM treatment fell to zero soon after this.

Conclusions:

Avidin is highly toxic to the black field cricket, demonstrating theefficacy of this protein as a means of controlling orthopteran pests.This suggests the use of avidin-expressing plants as a means ofcontrolling pests such as locusts and grasshoppers as well as crickets.

EXAMPLE 13 Toxicity of Artificial Diet Containing Streptavidin toNeonate Clover Root Weevil (Sitona lepidus) (Coleoptera: Curculionidae)and Neonate Argentine Stem Weevil (Listronotus bonariensis) (Coleoptera:Curculionidae)

Insects:

Eggs of both weevil species were obtained from field-collected adultsmaintained on white clover, Trifolium repens, (for Sitona lepidus) andryegrass, Lolium perenne, (for Listronotus bonariensis) foliage.

S. lepidus eggs were placed in Petri dishes on filter paper moistenedwith sterile distilled water and allowed to hatch at 25° C. To delayhatching until sufficient eggs had been laid for a trial, some eggs werestored for up to 24 days at 10° C., before being brought to the highertemperature for hatching.

L. bonariensis eggs were placed directly onto blocks of artificial dietin small plastic containers (4 mL autoanalyser cups), one larva per cup.

Streptavidin:

The streptavidin used in this trial was obtained fromCalbiochem-Novabiochem Corporation, and was a lyophilized solid. ProductNumber 189730. Lot Number B19870.

Diets:

An artificial diet (ASW diet) known to be suitable for rearing L.bonariensis (Malone and Wiley, 1990) was modified by omitting biotinfrom the recipe and used in the streptavidin trials for both weevilspecies.

To test the diet's suitability for S. lepidus trials, some of the firstneonate S. lepidus larvae obtained were placed onto blocks of unmodifiedASW diet (with biotin) for three days prior to being used in the firstreplicate of the streptavidin feeding trial. Other larvae in this trialhad been maintained for the first three days of life on either washedclover roots or a second artificial diet, which also contained biotin(porina diet) (Burgess et al. 1993). As larval feeding was observed onlyon ASW diet, a “biotin-free” version this diet was used in thesubsequent streptavidin trial. Neonate S. lepidus larvae used in thesecond and third replicates had had no previous exposure to diets ornatural foods containing biotin, but were used directly in thestreptavidin trial.

Trial Designs:

S. lepidus

For the S. lepidus streptavidin trial, neonate or 3-day-old larvae weretransferred individually to wells of microtitre trays containing“biotin-free” ASW diet. Three replicates were set up, each consisting of100 larvae receiving a streptavidin treatment and 100 larvae ascontrols. For the ‘treatment’ group, 0.9 mg/mL streptavidin (55 μM) wasblended thoroughly into the diet before it was dispensed into the wells.Control larvae received “biotin-free” ASW diet without any additive.Microtitre trays containing the diets were first covered by an ironed-onlayer of Mylar® film. Larvae were then introduced into each well viaslits cut in the Mylar® and then sealed in by a second covering, thistime of Frisk® adhesive film. They were observed daily for signs offeeding, burrowing and movement.

After 11 to 25 days, the films were removed from the trays and eachlarva was picked out of the diet and placed individually on a small cutblock of the same diet in an autoanalyser cup (4 mL). Any deaths wererecorded at this time and at approximately weekly intervals thereafteruntil the end of the experiment (94 days for Replicate 1:80 days forReplicate 2 and 78 days for Replicate 3).

L. bonariensis

For the L. bonariensis streptavidin trial, “biotin-free” ASW diet wasmade up as before, with the addition of 0.9 mg/mL streptavidin (55 μM)for the treatment group. Three replicates, each consisting of 31streptavidin-fed and 31 control larvae, were set up. For these weevils,each egg was placed directly on a cut block of the appropriate diet inan autoanalayser cup (4 mL) and sealed with its plastic lid. They wereexamined daily to observe larval hatching, feeding, burrowing ormovement. At approximately weekly intervals the containers were opened,the diet block teased apart and larval deaths recorded. Fresh diet ofthe same type was provided when required. The experiment was ended after51 days, when many of the control insects were still alive.

Results:

S. lepidus

In each replicate, larval survival was significantly lower for weevilsfeeding on diet with 55 μM streptavidin added than for the controlweevils (P<0.001, log-rank tests to compare survival curves (Kalbfleischand Prentice, 1980)). FIG. 38 shows the survival curves for allreplicates combined. Many of the larvae in the streptavidin treatedgroup appeared to have died during or immediately after a larval moult.Dead larvae often had a soft, transparent head and the darker discardedhead capsule attached to the rear of the insect.

Table 7 shows the median survival times for each group of weevils.Weevils in Replicate 1 had better survival than those in Replicates 2and 3. This may be due to the Replicate 1 weevils receiving eitherclover roots or ASW or porina diet with biotin aided for three daysbefore the start of the trial. In each case however, larvae treated withstreptavidin died significantly sooner than the control larvae.

Control survival was poorer than might be expected for weevils in thefield and only four control weevils developed into adults before the endof the experiment, probably because ASW diet was not the ideal mediumfor rearing this insect. No adults emerged among the clover root weevilsfed streptavidin.

L. bonariensis

In each replicate, larval survival was significantly lower for theweevils fed streptavidin than for the controls (P<0.001, log-rank teststo compare survival curves). FIG. 39 shows the survival curves for datafrom the three replicates combined. As with the clover root weevils,Argentine stem weevil larvae that had received streptavidin appeared tohave died during the moulting process and discarded head capsules werefound adhering to the rear ends of dead larvae.

Conclusions:

Streptavidin has significant toxicity to the larvae of two plant-eatingweevils, the clover root weevil. S. lepidus, and the Argentine stemweevil, L. bonariensis. This suggests that pasture plants expressingbiotin-binding proteins in the roots or stems could be protected fromattack by these pests.

TABLE 7 Median survival times for S. lepidus larvae (days). 95%confidence intervals in brackets Replicate 1 Replicate 2 Replicate 3Streptavidin Treatment 14 (11-15) 4 (4-6)   4 (3-7) Control 24 (21-29) 8(4-18) 16 (11-25)

EXAMPLE 14 Feeding Trials with Adult Clover Root Weevils (Sitonalepidus) (Coleoptera: Curculionidae) fed with Avidin-painted Clover(Trifolium repens) Foliage

Methods:

Adult Sitona lepidus were collected from a field at Ruakura AgriculturalResearch Centre, Hamilton, New Zealand, using a suction-poweredinsect-collecting device. They were then placed individually in clearplastic 30 mL containers (“Coulter cups”) with vented lids, eachcontaining a single painted leaf of white clover (Trifolium repens) withits stem embedded in about 10 mL of 0.4% agar in the bottom of the cup.This kept the leaf turgid for several days, while providing the weevilwith a solid surface to walk on.

The avidin used in this trial was a Calbiochem® product, purchased fromCalbiochem-Novabiochem Corporation, La Jolla, Calif. 92039. It waslyophilized avidin from egg white, Lot 276992.

As the upper surfaces of clover leaves are very hydrophobic, and S.lepidus adult weevils typically consume the entire leaf, only theundersides of the leaves were painted. The following solutions wereapplied with a small sable brush:

-   -   1. Controls were painted with 0.1% (v:v) BondXtra® (a wetting,        spreading and sticking agent) at a rate of 80 μl solution per g        of leaf (fresh weight).    -   2. “Low” avidin treatment leaves were painted with a 5 mg/mL        avidin solution in 0.1% BondXtra® at the same rate as above.        This rate approximates a leaf expressing 26 μM avidin.    -   3. “High” avidin treatment leaves were painted with a 10 mg/mL        avidin solution in 0.1% BondXtra® at the same rate as above.        This rate approximates a leaf expressing 52 μM avidin.

Between 15 and 18 adult weevils were placed on control leaves. 16 to 18on low avidin-painted leaves and 16 to 18 on high avidin-painted leaves.The experiment was replicated three times (total of 149 weevils).

Weevils were examined and deaths recorded every weekday until allweevils had died.

Results:

There were no significant differences among the survival curves foradult S. lepidus fed clover leaves painted with two doses of avidin orwith a control solution without avidin (FIG. 40) (log-rank test,Kalbfleisch and Prentice, 1980).

Conclusions:

Avidin is not toxic to adult clover root weevils, S. lepidus, whenpainted onto clover leaves at approximately 26 or 52 μM. It is thusunlikely that transgenic clover plants expressing avidin at these levelswill have toxicity to the adult stage of this weevil.

EXAMPLE 15 Feeding Trials with Adult Honeybees, Apis mellifera(Hymenoptera: Apidae), and Artificial Diet Containing Avidin

Method:

Young adult honeybees were collected as they emerged from frames ofcapped bee brood taken from hives kept at our apiary in Auckland, NewZealand.

The avidin used in this trial was a Calbiochem® product, purchased fromCalbiochem-Novabiochem Corporation, La Jolla, Calif. 92039. It waslyophilized avidin from egg white, Lot 276992.

Bees were assigned randomly to wooden cages (9×8×7 cm) with mesh on twosides, 30 bees per cage. Each cage was fitted with two gravity feeders,one containing water and the other sugar syrup (60% w:v sucrosesolution). These were replenished as necessary during the experiment.

Each cage was also provided with a small cup containing a mixture ofbee-collected pollen (1 part) and sugar candy (2 parts) (candy recipe:Ambrose, 1992) to which avidin had been added at two differentconcentrations. One group of cages was supplied with pollen/candy towhich 0.1 mg avidin per g of pollen had been added (equivalent toapproximately 6.7 μM avidin) and a second group was supplied with amixture containing 0.3 mg avidin per g of pollen (equivalent toapproximately 20 μM avidin). A third set of bees (controls) receivedpollen/candy without additive. The trial was replicated four times. i.e.a total of 12 cages of bees.

To measure consumption of the pollen/candy food by the bees, each cupwas weighed at the start of the experiment and again at Days 8 and 14.Each cage was checked daily for bee deaths.

Results:

There were no significant differences in the mean quantities ofpollen/candy consumed by the three groups of bees (ANOVA) over the first8 days of exposure to the foods, between Days 8 and 14, or over theentire 14-day period (FIG. 41).

Comparisons of survival curves using log-rank tests (Kalbfleisch andPrentice, 1980) showed that bees fed the higher dose of avidin hadsignificantly better survival (P<0.002) than those fed the lower dose.Control bee survival was intermediate between, and did not differsignificantly from, that of bees fed either avidin dose (FIG. 42).

Conclusions:

Adult honeybees readily consume pollen/candy mixtures containingapproximately 6.7 or 20 μM avidin and, when compared with control bees,their survival is unaffected by this consumption. This suggests that ifbiotin-binding proteins are expressed at these levels in pollen fromplants modified to contain these genes, then young adult bees will notbe repelled or harmed by such pollen.

EXAMPLE 16 Feeding Trials with Slugs (DerocerasReticulatum)(Stylommatophora: Agriolimacidae) and Snails (Cantareusaspersus) (Stylommatophora: Helicidae) Fed with Avidin Painted ontoLettuce (Latuca saliva) Foliage

Methods:

Snails and slugs were collected from local gardens (Auckland, NewZealand), weighed and placed in groups in sealed plastic containers(220×160×40 mm) with organically-grown lettuce leaves coated thoroughlywith one of the following treatments:

-   1. Controls were painted with 0.1% (v:v) BondXtra® (a wetting    spreading and sticking gent) only;-   2. 4.8 μM avidin treatment leaves were painted with an avidin    solution in 0.1% BondXtra® that delivered 75 μg of avidin per g    fresh weight of lettuce;-   3. 19.2 μM avidin treatment leaves were painted with an avidin    solution in 0.1% BondXtra® that delivered 300 μL of avidin per g    fresh weight of lettuce.

Each container was checked daily for deaths, the interior sprayed withwater mist and the painted lettuce replenished as necessary. At the endof the experiment (after 51 days) all surviving animals were weighed.

Snails:

Snails were individually identified with a number written on theirshells with permanent marker pen. Two containers of ten snails each wereset up for each treatment (Le. 3 treatments×2 containers×10 snails=60snails total). Each snail was weighed at the beginning of the experimentand the survivors also weighed at the end.

Slugs:

Five containers, each containing five slugs, were set up for each of thethree treatments (i.e. 3 treatments×5 containers×5 slugs=75 slugstotal). As slugs could not be individually marked, all five from eachcontainer were weighed together at the be beginning of the experiment.Surviving slugs were weighed individually at the end of the experiment.

Results:

Snails:

The three groups of snails used in the experiment had similar initialweights (ANOVA, FIG. 43). All snails grew during the 51-day experimentand there were no significant differences in final weights among thethree groups (ANOVA, FIG. 43).

Few snails died during the experiment (FIG. 44). There were nosignificant differences in mean snail longevity among the three groups(ANOVA, a 51-day longevity was assumed for all snails alive at the endof the experiment. i.e. an underestimate).

Slugs:

Initial weights of slugs were also similar across the three groups, buteither lettuce must have been a poor diet for them or the conditions intheir containers did not favour their development, because all slugslost weight during the experiment (FIG. 45). There were no significantdifferences attributable to the treatments in initial or final mean slugweights (ANOVA).

Slug survival, particularly among the controls, was also poor underthese experimental conditions (FIG. 46). In fact slugs on lettucepainted with either 4.8 μM or 19.2 μM avidin had significantly greatermean longevity than the control slugs (ANOVA, P=0.040, assuming allsurviving slugs at the end of the experiment had a longevity of 51days).

Conclusions:

Avidin had no effect on snail growth or survival when applied to theirlettuce leaf food at 4.8 μM or 19.2 μM for a period of 51 days.

Slug results were confounded by poor growth of all slugs and poorsurvival of controls during the trial. However, avidin had no obvioustoxicity to these invertebrates over a 51-day period of receivinglettuce painted with 4.8 μM or 19.2 μM of this protein.

EXAMPLE 17 Evaluation of Resistance of Tobacco (Nicotiana tabacum)Plants Expressing Avidin to Three Species of Root-knot Nematodes

Methods:

Plants:

Tobacco seedlings were germinated either from non-transgenic (NT) seedor from seed collected from three independent selfed originaltransformant plants (PLA2/1, PLA2/4 and PLA2/24).

Avidin Expression Levels:

Twenty five control and 25 transgenic seedlings were transferredindividually to 60-mm-diameter plastic pots of peat based porting mixand left to grow for a week before leaf samples were taken for ELISAanalysis of gene expression (Example 5).

Avidin levels in both roots and leaves were measured earlier in 14transgenic seedlings (from selfed independent oringinal transformantsPLA2/7, PLA2/9 and PLA2/13) and two non-transgenic plants. Levels ofavidin varied between 0 and 2.23 μM in roots and 0 and 16.84 μM inleaves. There was a linear correlation between leaf and root avidinlevels in individual plants (n=16, R²=0.716). Leaf avidin levels weresubsequently used to select experimental material since it is notpossible to harvest and measure root material prior to assay. Biotinconcentrations in these plants were independent of avidin expression,being 0.05 μM in root tissue and 0.7 μM in leaves.

Nematodes:

Twenty highly expressing PLA2 plants and 20 non-transgenic plants werere-potted into 100-mm-diameter pots and a week later inoculated with asuspension 4000 eggs of root-knot nematodes injected into holes aroundthe roots (method described in Sasser and Carter 1985). The nematodespecies used were Meloidogyne javanica, Meloidogyne hapla andMeloidogyne incognita. Control plants were injected with water. Thus,the design was 3 nematode species+1 control=4 inoculation types X2 Genecategories X5 replicates=40 pots.

After seven weeks, roots were washed free of potting mix and the gallscounted. Roots and galls were then crushed with a small roller andextracted in chlorine solution to free the eggs, which were sieved outand counted.

Results:

The levels of avidin in the transgenic plants were 11.4±6.8 μM (range2.4-26.05). Even at the lowest avidin level a six-fold molar excess ofavidin over biotin can be calculated. There were no significantdifferences between means of gall and egg counts for each of the threeroot-knot nematode species (P>0.10) (ANOVA. Sokal and Rohlf 1969) (Table8). No galls were seen on sham inoculated plants.

Conclusion:

Transgenic tobacco expressing high levels of avidin in root tissue isnot resistant to root-knot nematode attack.

TABLE 8 Number of eggs laid and galls formed on tobacco roots by threespecies of nematodes. M. javanica M. hapla M. incognita Mean Mean MeanMean Plant Galls (s.e.) Eggs (s.e.) Plant Galls Mean Eggs (s.e.) PlantGalls (s.e.) Eggs Mean NT NT NT  1 209 0  8 208 87  9 28 0  2 185 63 10249 0 15 14 41  3 127 0 14 256 22 21 22 20 13 222 0 16 251 0 22 17 34 17112 171 56 24 20 247 242 0 22 23 51 26 0 19  (22) (15)  (9) (17)  (7) (8) PLA2/ PLA2/ PLA2/  1/6 139 17 24/9 149 34  1/7 30 16  4/1 175 4224/11 216 26  4/8 21 0 24/1  74 16 24/12 197 65  4/14 22 0 24/4  89 2724/14 189 115 24/5 16 25 24/7 127 121 0 20  4/24 277 206 62 60 24/8 3224 18 12  (18)  (7)  (21) (16)  (3)  (5)

EXAMPLE 18 Combined Toxic Effects of Avidin Expressed in Tobacco(Nicotiana tabacum) Leaves Painted with Either a Protease Inhibitor or aBt Insecticidal Protein to Larval Helicoverpa armigera (Lepidoptera:Noctuidae): Bt and Avidin act Synergistically

Constructs:

Control lines:

Non-transformed control plants:

Two hundred and forty one plants (NT 201-NT 441) were grown from seedsproduced by selfed NT plant 11 which was used in the trial described inExample 8.

Avidin-expressing lines:

Selfed T₂ avidin-expressing generation

Tobacco plants were grown from seeds collected from three of the plantsused in the trial described in Example 9. These parent plants wereselfed (self-fertilised) and were the T₁ offspring of plants from theoriginal transformant (T₀) plant lines PLA2/7, PLA2/9 and PLA2/13 usedin the trial described in Example S. The plants used in this trial werethus second-generation (T₂) selfed plants derived from plants which hadbeen transformed with the avidin gene with a PPI-I targeting sequence(Example 2).

Ninety eight plants from the PLA2/7 #18 line, 99 from the PLA2/9 #24line and 126 from the PLA2/13 #22 line were grown for the experiment.

On each plant used in the experiment, Leaf 1 was designated as theuppermost (youngest) leaf which was 8 cm or more in length from leaf tipto junction of leaf base with the petiole. The leaves below Leaf 1 wereassigned numbers consecutively down the plant Leaves 1, 2 and 3 wereused for H. armigera feeding, as previous experiments had shown larvaegrow best on young leaves.

Insecticidal Proteins:

Two purified insecticidal proteins were painted onto tobacco foliage inthis experiment:

-   -   Bacillus thuringiensis insecticidal protein, Cry1Ba Activated        Cry1Ba toxin was obtained from a large-scale fermentation of B.        thuringiensis Bt4412, purified and cleaved according to the        method described by Simpson et al. (1997).    -   Protease inhibitor, aprotinin, obtained from Intergen® Company,        Canada/USA (Product No. 7105, Lot No. NT59808).        Insects:

H. armigera were obtained from a laboratory colony reared on artificialdiet as described in McManus and Burgess (1995) and established frommoths collected in Christchurch, New Zealand.

Neonate H. armigera larvae were placed on artificial diet for 48 hfollowing emergence from eggs. These late first instar larvae were thenplaced on tobacco leaves as described below. Initial larval weight wasdetermined as the mean of the individual weights of a randomly chosensample of 54 of the larvae used in the trial.

Determination of Avidin Expression Levels:

Before commencing the experiment, a whole leaf sample comprising a leafof at least 8 cm in length was taken from the 263 of the 323avidin-expressing plants which had grown the best over an eight weekperiod. Eighty nine PLA2/7 #18 plants, 71 PLA2/9 #24 plants and 103PLA2/13 #22 plants were tested for avidin expression level using theELISA assay described in Example 5. The plants were then rankedaccording to avidin expression level. Plants from the top of the tablewere then used in treatments requiring “high” expressors and those fromthe bottom of the table used where “low” expressors were required.

Trial Design:

Larvae were subjected to nine different treatments to test the effectsof avidin, aprotinin and Cry1Ba separately and in two-way combinations.Each leaf was weighed before painting, and all solutions were applied ata rate of 100 μL solution per g of fresh leaf. To ensure leaves remainedturgid, the petiole of each cut leaf was immersed in a setting solutionof 0.4% w:v agar in a 30 mL coulter cup.

Treatments:

-   -   Control tobacco leaves painted with a control solution of 0.1%        (v:v) BondXtra® (a wetting, spreading and sticking agent).    -   Control tobacco leaves painted with a 2 mg/ml solution of        aprotinin in 0.1% (v:v) BondXtra® at the same rate as above. If        tobacco leaves are about 2% protein, then this rate approximates        a leaf expressing aprotinin as 1% of total soluble protein.    -   Control tobacco leaves painted with a 1 mg/ml solution of Cry1Ba        in 0.1% (v:v) BondXtra® at the same rate as above. If tobacco        leaves are about 2% protein, then this rate approximates a leaf        expressing Cry1Ba as 0.5% of total soluble protein.    -   Tobacco leaves expressing avidin at a “low” level (see below)        and painted with 0.1% (v:v) BondXtra®    -   Tobacco leaves expressing avidin at a “low” level and painted        with a 2 mg/ml solution of aprotinin in 0.1% (v:v) BondXtra®    -   Tobacco leaves expressing avidin at a low” level and painted        with a 1 mg/ml solution of Cry1Ba in 0.1% (v:v) BondXtra®    -   Tobacco leaves expressing avidin at a “high” level (see below)        and painted with 0.1% (v:v) BondXtra®    -   Tobacco leaves expressing avidin at a “high” level and painted        with a 2 mg/ml solution of aprotinin in 0.1% (v:v) BondXtra®    -   Tobacco leaves expressing avidin at a “high” level and painted        with a 1 mg/ml solution of Cry1Ba in 0.1% (v:v) BondXtra®

The ranges of avidin expression levels in the plants used were asfollows:

-   Treatment 4 (“low”): 2.12-5.27 μM-   Treatment 5 (“low”): 2.62-5.30 μM-   Treatment 6 (“low”): 3.62-5.24 μM-   Treatment 7 (“high”): 12.95-21.27 μM-   Treatment 8 (“high”): 12.90-21.00 μM-   Treatment 9 (“high”): 14.18-18.10 μM

Ten larvae were placed on the underside of each treated leaf inside a300×210×80 mm plastic storage box lined with paper towels and with asnap-on lid. Three replicate boxes were set up for each treatment, i.e.27 boxes in total, 30 larvae per treatment (two of the treatments wereinadvertently given 31 larvae). Larvae and leaves were checked daily,and leaves were replaced with identically treated fresh leaves fromsimilar plants as necessary so that larvae could feed ad libitum.

The experiment was conducted in a controlled temperature room at 30±1°C. and 60% relative humidity, with a 16:8 h light:dark cycle.

Larval deaths were recorded on Day 2 and daily thereafter for 14 days oruntil or all had died if this occurred earlier. Larvae were weighed onDays 3, 6, 8, 10 and 12. Once larvae had begun to pupate in anytreatment, larvae in that treatment were no longer weighed.

Results:

Survival curves for H. armigera in the nine different treatment groupsare shown in FIG. 47. Log-rank tests (Kalbfleisch and Prentice, 1980)were used to compare median survival in the different treatments. Theonly treatment which did not reduce median survival time compared withcontrol survival was that using aprotinin-painted control leaves.

The four treatments using leaves expressing avidin at both high and lowlevels, with and without aprotinin painted on, killed all larvae within13 days. Death often occured during early larval instar moulting.Survival on all these treatments was significantly reduced in comparisonwith survival on control leaves with and without aprotinin (ANOVA P25<0.001) (Payne etal. 1993). Median survival times on these fouravidin-expressing treatments did not differ significantly from eachother. Thus the effect on median larval survival of the combination ofavidin expression and aprotinin was equivalent to the effect of avidinexpression alone. However, closer examination of the survival curves forthe “low avidin” and the “low avidin with aprotinin” reveals that theydiverge between days 8 and 12. The proportion of larvae alive on the“low avidin with aprotinin” treatment is significantly lower on days 9,10 and 11 (ANOVA P<0.05). This demonstrates that avidin can be combinedwith a protease inhibitor to produce a more toxic effect on larvae, eventhough the effect of the protease inhibitor alone may be subtle.Additionally, there is no suggestion of antagonism between the two typesof resistance protein.

The three treatments in which Cry1Ba was painted onto the leaves killedall larvae within four days. Larvae feeding on high and lowavidin-expressing leaves painted with Cry1Ba died significantly fasterthan those feeding on Cry1Ba-painted control leaves (ANOVA P<0.001). Theeffects on mean larval survival of both the high avidin/Cry1Ba and thelow avidin/Cry1Ba combination treatments were greater than the sum ofthe effects of the high or low avidin expression alone and Cry1Bapainting alone. Thus synergistic effects were observed with thesecombinations.

Growth rates and biomass were plotted for larvae on all the non-Cry1Batreatments. Larvae feeding on control plants painted with the controlsolution or the aprotinin solution grew and accumulated biomassexponentially, while those on all treatments expressing avidin at highor low levels failed to grow or accumulate substantial biomass (FIGS. 48And 49). Because of the powerful effects of the avidin alone, it was notpossible to measure any more subtle effects that the combination withaprotinin may have had on these two parameters.

Conclusions:

Synergistic toxic effects on H. armigera larvae were observed withcombinations of avidin-expressing tobacco leaf and the Bt insecticidalprotein. Cry1Ba.

This suggests strongly that plants containing chimeric genes andexpressing both avidin and Bt will be highly effective in protecting theplants from pest attack. It is likely that such plants will be moretoxic than those expressing either protein singly.

Avidin at both high and low expression levels when combined withaprotinin was as effective as avidin expression alone in killing larvaeand preventing growth and biomass accumulation. During the latter partof the experiment, larval death was greater in the combinedaprotinin/low avidin treatment than in the low avidin treatment alone.This demonstrates the possibility that additive or synergistic effectscould occur between avidin or streptavidin and a protease inhibitorwhich reduces larval growth and survival. The absence of anyantagonistic effects between the biotin binding protein and the proteaseinhibitor shows the compatibility of these two types of resistancefactor.

It is likely that plants expressing avidin together with a secondeffective insecticidal protein employing a different mode of action willnot only have greater toxicity, but also more durable resistance to pestattack than plants expressing or containing a Bt protein, a proteaseinhibitor or another type of pest resistance factor on its own.

REFERENCES

-   Argarana C. E., Kuntz, I. D., Birkens, Axel R., and Cantor C. R.    (1986). Molecular Cloning and Nucleotide Sequence of the    Streptavidin Gene Nucleic Acids research 14: 1871-1882.-   Bayer E. A. Ben-Hur H., Wilchek M. (1990) Isolation and properties    of streptavidin. Methods in Enzymology 184: 80-89.-   Bednarek S Y, Wilkins T. A., Dombrowski J. E., Raikhel N V (1990) A    carboxyl-terminal propeptide is necessary for proper sorting of    barley lectin to vacuoles of tobacco. Plant Cell 1990 2:1145-1155.-   Beuning L. L. Spriggs T. W., and Christeller J. T. (1994) Evolution    of the Proteinase Inhibitor I Family and Apparent Lack of    Hypervariability in the Proteinase Contact Loop. J. Mol. Evol. 39    (6): 644-654.-   Boulter, D. (1993). Review Article Number 86. Insect Pest Control by    Copying Nature using Genetically Engineered Crops. Phytochemistry    34: 1453-1466.-   Broodryk, S. W. 1971. Ecological investigations on the potato tuber    moth Phthorimaea operculella (Zeller) (Lepidoptera: Gelechiidae).    Phytophylactica 3: 73-84.-   Burgess, E. P. J., Main, C. S., Stevens, P. S., Gatehouse, A. M. R.,    Christeller, J. T. and Laing, W. A. 1993. Protease inhibitors active    against porina caterpillar (Wiseana cervinata). Proc. 6^(th)    Australasian Grassl. Invert. Ecoli. Conf. 1993 (R. A., Prestidge,    Ed.). AgResearch, Hamilton, New Zealand, 331-339.-   Chrispeels, M. J. (1991). Sorting of Proteins in the Secretory    System. Annu. Rev. Plant Physiol. Plant Mol. Biol. 42: 21-53.-   Chrispeels M. J, Raihkel N V. Short peptide domains target proteins    to plant vacuoles. Cell. 1992 Feb. 21; 68(4): 613-616.-   Cleveland, T. E. Thornburg, R. W., and Ryan, C. A. (1987). Molecular    Characterization of Wound-inducible Inhibitor I Gene from Potato and    the Processing of its mRNA and Protein. Plant Mol. Biol. 8: 199-207.-   Dadd, R. H. (1985). Nutrition:Organisms In: Comprehensive Insect    Physiology, Biochemistry and Pharmacology (Kerkut G. A. and    Gilbert, L. L.) Pergamon Press, NY Vol 4, p 313-390).-   Ditta G., Stanfield S., Corbin D., and Helinski D. R. (1980) Broad    Host Range DNA Cloning System for Gram-Negative Bacteria:    Construction of a Gene Bank of Rhizobium meliloti. Proc Natl Acad    Sci USA 77 (12): 7347-7351.-   Donson J, Kearney C M, Hilf M E, Dawson W O (1991); Systematic    expression of a bacterial gene by a tobacco mosaic virus-based    vector. Proc Natl. Acad. Sci. USA., 88:7204-8.-   Drombrowski J E, Raikhel N V. Protein targeting to the plant    vacuole—a historical perspective. Braz J. Med. Biol. Res. 1996    April; 29(4): 413-430.-   Gleave A. P. (1992) A Versatile Binary Vector System with a T-DNA    Organisational Structure Conducive to Efficient Integration of    Cloned DNA into the Plant Genome. Plant Mol. Biol. 20 (6):    1203-1207.-   Gleave, A. P., Mitra, D. S., Markwick, N. P., Morris. B. A. M. and    Beuning, L. L. 1998. Enhancing the expression of the Bacillus    thuringiensis cry9Aa2 gene in transgenic plants by nucleotide    sequence modification. Molecular Breeding 4: 459-472.-   Gope M. L., Keinanen R. A., Kristo P. A., Conneely O. M., Beattie W.    G., Zarucki-Schulz T., O'Malley B. W. and Kulomaa M. S. (1987)    Molecular Cloning of the Chicken Avidin cDNA. Nucleic Acids Res. 15    (8): 3595-3606.-   Graham, J. S., Pearce, G., Merryweather, J. Titani, K.,    Ericsson L. H. and Ryan. C. A. (1985). Wound-induced Proteinase    Inhibitors from Tomato Leaves. J. Biol. Chem. 260: 6561-6564.-   Holwerda B C, Padgett H S. Rogers J C (1992) Proaleurain vacuolar    targeting is mediated by short contiguous peptide interactions.    Plant Cell 4:307-318.-   James, C. and A. F. Krattiger (1996). Global Review of the Field    Testing and Commercialisation of Transgenic Plants, 1986 to 1995:    The First Decade of Crop Biotechnology. International Service for    the Acquisition of Agri-Biotech Applications (ISAAA) Briefs No.    1.11) ISAAA: Ithaca, N.Y. pp 31.-   Joshi R L, Joshi V, Ow D W (1990) BSMV genome mediated expression of    a foreign gene in dicot and monocot plant cells. EMBO J. 9:2663-9.-   Kalbfleisch, J. D. and Prentice, R. L. 1980. Statistical Analysis of    Failure Time Data. John Wiley, New York.-   Keinanen R A, Wallen M J, Kristo P A, Laukkanen M O, Toimela T A,    Helenius M A, Kulomaa M S Molecular cloning and nucleotide sequence    of chicken avidin-related genes. 1-5. Eur. J, Biochem. 220:615-621    (1994).-   Keller, M., Sneh, B., Strizhov, N., Prudovsky, E., Regev, A., Koncz,    C., Schell, J., Zilberstein, A. (1996): Digestion of delta-endotoxin    by gut proteases may explain reduced sensitivity of advanced instar    larvae of Spodoptera littoralis to CryIC. Insect Biochem. Mol. Biol    26: 365-73.-   Kirsch T, Paris N, Butler J M, Beevers L, Rogers J C. Purification    and initial characterization of a potential plant vacuolar targeting    receptor. Proc. Natl. Acad. Sci. USA. 1994 Apr. 12; 91(8):    3403-3407.-   Malone, L. A. and Wigley, P. J. 1990. A practical method for rearing    Argentine stem weevil, Listronotus bonariensis (Coleoptera:    Curculionidae) in the laboratory. N.Z Entomol. 13, 87-88.-   Mariani, C., Goldberg R. B., Leemans, J. (1991), Engineered male    sterility in plants. Symp. Soc. Exp. Biol. 45:271-9.-   Martilla A T, Airenne K J, Laitinen O H, Kulik T. Bayer E A, Wilchek    M, Kulomaa M S. Engineering of chicken avidin: a progressive series    of reduced charge mutants. FEBS Letters 441: 313-317 (1998).-   Matsuoka, K. Matsumoto, S., Hattori, T., Machida, Y., and    Nakamura, K. (1990) Vacuolar Targeting and Posttranslational    Processing of the Precursor to the Sweet Potato Tuberous Root    Storage Protein in Heterologous Plant Cells. J. Biol. Chem. 265:    19750-19757.-   Meisner, J., K. R. S. Ascher, and D. Lowie. 1974. Phagostimulants    for the larva of the potato tuber moth, Gnorimoschema operculella    Zell. Z. Angew. Entomol. 77: 77-106.-   Michelmore, R, Marsh, E., Seely, S., and Benoit, L. (1987).    Transformation of lettuce (Lactuca sativa) mediated by Agrobacterium    tumefaciens. Plant Cell Reports 6:439-442.-   Miller, D. W., et al., in Genetic Engineering (1986) Setlo W, J K et    al., Eds, Plenum Publishing, Vol 8: pages 277-297).-   Moore D. S., and Michael S. F. (1995) Mutagenesis of Amplified DNA    Sequences Using Ampligase Thermostable DNA Ligase. Epicentre Forum 2    (4): 4-5.

Morgan, T. D., B. Oppert, T. H. Czapala, and K. J. Kramer (1993). Avidinand Streptavaidin as Insecticidal and Growth Inhibiting DietaryProteins. Entomol. exp. appl. 69: 97-108.

-   Murray C. and Christeller J. T. (1994) Genomic Nucleotide Sequence    of a Proteinase Inhibitor II Gene. Plant Physiol. 106: 1681.-   Nakamura, K., and Matsuoka, K. (1993) Protein Targeting to the    Vacuole in Plant Cells. Plant Physiol. 101: 1-5.-   Neuhaus J M, Sticher L, Meins F Jr, Boller T (1991) A short    C-terminal sequence is necessary and sufficient for the targeting of    chitinases to the plant vacuole. Proc Natl Acad Sci USA    88:10362-10366.-   Nielsen K J, Hill J M, Andersom M A, Craik D J. Synthesis and    structure determination of NMR of a putative vacuolar targeting    peptide and model of proteinase inhibitor from Nicotiana alata.    Biochemistry. 1996 Jan. 16; 35(2): 369-378.-   Rusch S L, Kendall D A. Protein transport via amino-terminal    targeting sequences: common themes in diverse systems. Mol. Membr.    Biol. 1995 October; 12(4): 295-307.-   Saalbach G, Rosso M, Schumann U (1996) The vacuolar targeting signal    of the 2S albumin from Brazil nut resides at the C terminus and    involves the C-terminal propeptide as an essential element. Plant    Physiol 112:975-985.-   Sanchez-Serrano, J., Schmidt, R., Schell. J., and Willmitzer, L.    (1986). Nucleotide Sequence of Proteinase Inhibitor II Encoding cDNA    of Potato (Solanum tuberosum) and its Mode of Expression. Mol. Gen.    Genet. 203: 15-20.-   Schatz P J. (1993) Bio/Technology 11, 1138-1143-   Schroder M R, Borkhsenious O W, Matsuoka K, Nakamura K Raikhel N V.    Colocalization of barley lectin and sporamin in vacuoles of    transgenic tobacco plants. Plant Physiol. 1993 February; 101(2):    451-458.-   Seshagiri P B, Adiga P R (i987) Isolation and characterisation of a    biotin-binding protein from the pregnant-rat serum and comparison    with that from the chicken egg-yolk. Biochim Biophys Acta    916:474-481.-   Shao, Z., Cui. Y., Liu, X., Yi, H., Ji, J., Yu, Z. (1998):    Processing of delta-endotoxin of Bacillus thuringiensis subsp.    Kurstaki HD-1 in Heliothis armigera midgut juice and the effects of    protease inhibitors., J Invertebr. Pathol. 72: 73-81.-   Subramanian N, Adiga P R (1995) Simultaneous purification of    biotin-binding protein-I and -II from chicken egg yolk and their    characterization. Biochem J 308:573-577.-   Tague B W, Dickinson C D, Chrispeels M Y (1990) A short domain of    the plant vacuolar protein phytohemagglutinin targets invertase to    the yeast vacuole. Plant Cell 2:533-546.-   Thompson L. D. and Weber P. C. (1993) Construction and Expression of    a Synthetic Streptavidin-Encoding Gene in Escherichia coli. Gene    136: 243-246.-   Torres, C., Cantliffe, D. J., Laughner, B., Bieniek, M. Nagata, R.,    Ashraf, M. and R. J. Feri (1993). Stable transformation of lettuce    cultivar South Bay from cotyledon explants. Plant Cell, Tissue and    Organ Culture 34: 279-285.-   Turpen, T H. (1999) Tobacco mosaic virus and the virescence of    biotechnology. Philos Trans. R Soc. Lond. Biol. Sci., 354: 665-73,    1999).-   Vitale A, Chrispeels M J (1992) Sorting of proteins to the vacuoles    of plant cells. Bioassays 14:151-160.-   Von Heijne, G. (1983). Patterns of Amino Acids Near Signal-Sequence    Cleavage Sites. Eur. J. Biochem. 133: 17-21.

Walker-Simmons, M., and Ryan, C. A. (1977). Immunological Identificationof Proteinase Inhibitors I and II in Isolated Tomato Leaf Vacuoles.Plant Physiol. 60: 61-63.

-   Wilcheta M, Bayer EA. (eds) 1990. Avidin-Biotin Biotechnology.    Methods of Enzymology Vol 184.-   Zhang, X., and Conner, A. J. (1992). Genotypic effects on tissue    culture response of lettuce cotyledons. J. Genet and Breed 46:    287-290.

All references are incorporated herein by reference.

1. An isolated nucleic acid molecule comprising: (i) a first nucleicacid sequence that encodes a vacuole targeting polypeptide operablylinked to (ii) a second nucleic acid sequence that encodes a biotinbinding protein selected from the group consisting of: (a) avidin; and(b) streptavidin.
 2. The nucleic acid molecule of claim 1, wherein thevacuole targeting polypeptide is a potato proteinase inhibitor signalpolypeptide.
 3. The nucleic acid molecule of claim 2, wherein thevacuole targeting polypeptide is a potato proteinase inhibitor I signalpolypeptide.
 4. The nucleic acid molecule of claim 2, wherein thevacuole targeting polypeptide is a potato proteinase inhibitor II signalpolypeptide.
 5. The nucleic acid molecule of claim 1, wherein the biotinbinding protein is avidin.
 6. The nucleic acid molecule of claim 1,wherein the biotin binding protein is streptavidin.
 7. The nucleic acidmolecule of claim 6, wherein streptavidin is selected from the groupconsisting of: Core streptavidin, synthetic Core streptavidin, andSYNSAV.
 8. The nucleic acid molecule of claim 6, wherein thestreptavidin is encoded by the sequence set forth in SEQ ID NO:10. 9.The nucleic acid molecule of claim 1, wherein the vacuole targetingpolypeptide is a potato proteinase inhibitor I polypeptide and thebiotin binding protein is avidin.
 10. The nucleic acid molecule of claim1, wherein the vacuole targeting polypeptide is a potato proteinaseinhibitor II signal polypeptide and the biotin binding protein isstreptavidin.
 11. The nucleic acid molecule of claim 1, wherein thevacuole targeting sequence is an N-terminal targeting polypeptide. 12.The nucleic acid molecule according to claim 1, wherein said nucleicacid molecule is a DNA molecule.
 13. A vector comprising the nucleicacid molecule according to claim
 12. 14. A host cell transformed withthe vector according to claim
 13. 15. The host cell according to claim14, wherein said cell is a plant cell.
 16. A method for producing abiotin-binding protein, said method comprising the steps of: (a)culturing a host cell which has been transformed with a vectorcomprising the nucleic acid molecule according to claim 12 to produce anexpressed biotin-binding protein; and (b) recovering the expressedbiotin-binding protein.
 17. A method for producing a pest resistantplant, said method comprising transforming the plant genome with atleast one nucleic acid molecule according to claim 12, thereby producinga pest resistant plant.
 18. A transgenic plant that comprises thenucleic acid molecule according to claim
 12. 19. A transgenic plantexpressing pesticidally effective concentrations of the biotin-bindingprotein, wherein the plant comprises the nucleic acid molecule accordingto claim
 1. 20. A method for producing a biotin-binding protein, saidmethod comprising extracting avidin or streptavidin from a planttransformed with the nucleic acid molecule according to claim
 1. 21.Seed that is the product of the plant according to claim 18, wherein theseed comprises the nucleic acid molecule.